s pyogenes  (New England Biolabs)


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    Name:
    Monarch PCR and DNA Cleanup Kit
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    Monarch PCR and DNA Cleanup Kit 250 preps
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    t1030l
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    New England Biolabs s pyogenes
    Monarch PCR and DNA Cleanup Kit
    Monarch PCR and DNA Cleanup Kit 250 preps
    https://www.bioz.com/result/s pyogenes/product/New England Biolabs
    Average 92 stars, based on 2888 article reviews
    Price from $9.99 to $1999.99
    s pyogenes - by Bioz Stars, 2020-09
    92/100 stars

    Images

    1) Product Images from "Staphylococcus aureus Cas9 is a multiple-turnover enzyme"

    Article Title: Staphylococcus aureus Cas9 is a multiple-turnover enzyme

    Journal: RNA

    doi: 10.1261/rna.067355.118

    S. pyogenes Cas9 binds sgRNA with a higher affinity than SauCas9 and both form active, sgRNA-dependent complexes with comparable K 1/2 for sgRNA
    Figure Legend Snippet: S. pyogenes Cas9 binds sgRNA with a higher affinity than SauCas9 and both form active, sgRNA-dependent complexes with comparable K 1/2 for sgRNA

    Techniques Used:

    2) Product Images from "Novel ssDNA Ligand Against Ovarian Cancer Biomarker CA125 With Promising Diagnostic Potential"

    Article Title: Novel ssDNA Ligand Against Ovarian Cancer Biomarker CA125 With Promising Diagnostic Potential

    Journal: Frontiers in Chemistry

    doi: 10.3389/fchem.2020.00400

    Serum stability profiling: (A) PCR amplified DNA and (B) its single-stranded form before amplification, after treatment with 50%v/v normal human female serum. Effect of different salt concentrations on aptamer-CA125 binding: (C) negative controls and (D) treated aptamer (lane 1: ladder, lane 2: 0.2 M NaHCO 3 with 0.5 M NaCl, lane 3: 100 mM NaCl and 5 mM MgCl 2 , lane 4: milli-Q water as positive control; (E) Binding—saturation curve for determination of K D . The original gel images have been provided in Figures S4, S5 .
    Figure Legend Snippet: Serum stability profiling: (A) PCR amplified DNA and (B) its single-stranded form before amplification, after treatment with 50%v/v normal human female serum. Effect of different salt concentrations on aptamer-CA125 binding: (C) negative controls and (D) treated aptamer (lane 1: ladder, lane 2: 0.2 M NaHCO 3 with 0.5 M NaCl, lane 3: 100 mM NaCl and 5 mM MgCl 2 , lane 4: milli-Q water as positive control; (E) Binding—saturation curve for determination of K D . The original gel images have been provided in Figures S4, S5 .

    Techniques Used: Polymerase Chain Reaction, Amplification, Binding Assay, Positive Control

    3) Product Images from "A fly model establishes distinct mechanisms for synthetic CRISPR/Cas9 sex distorters"

    Article Title: A fly model establishes distinct mechanisms for synthetic CRISPR/Cas9 sex distorters

    Journal: bioRxiv

    doi: 10.1101/834630

    (A) Assay to detect CRISPR/Cas9-mediated cleavage in vitro . A typical region of the Muc14a gene containing at least 2 binding sites for each of the gRNAs: Muc14a _3, Muc14a_4 , Muc14a_5 and Muc14a_6 (top). The PCR amplified DNA fragment was used as a digestion target for Cas9/gRNA cleavage reactions in vitro (bottom). Reactions were run on a gel to detect cleavage. A control without gRNA was included. (B) Analysis of combinations of gRNAs and Cas9 sources for X-shredding. Average male frequencies in the F1 progeny are shown for each parental genotype with a single copy of βtub85Dtub85D-cas9 transgene (1X), two copies of βtub85Dtub85D-cas9 transgene (2X) or one copy of nos-cas9 (grey bars). All lines were crossed to wild type w individuals. The reciprocal cross (female ctrl) or heterozygote βtub85Dtub85D-cas9/ + or nos-cas9/ + without gRNA (no gRNA) were used as control. The black arrow indicates gRNAs in the multiplex array and the red dotted line indicates an unbiased sex-ratio. Crosses were set as pools of males and females or as multiple male single crosses in which case error bars indicate the mean ± SD for a minimum of ten independent single crosses. For all crosses n indicates the total number of individuals (males + females) in the F1 progeny counted. (C) Developmental survival analysis of the F1 progeny of Muc14a_6/βtub85Dtub85D-cas9 males crossed to w females compared to w and βtub85Dtub85D-cas9/ + control males crossed to w females. n indicates the number of individuals recorded at every developmental stage (males + females) in the F1 progeny. Bars indicate means ± SD for at least ten independent single crosses. Statistical significance was calculated with a t test assuming unequal variance. ** p
    Figure Legend Snippet: (A) Assay to detect CRISPR/Cas9-mediated cleavage in vitro . A typical region of the Muc14a gene containing at least 2 binding sites for each of the gRNAs: Muc14a _3, Muc14a_4 , Muc14a_5 and Muc14a_6 (top). The PCR amplified DNA fragment was used as a digestion target for Cas9/gRNA cleavage reactions in vitro (bottom). Reactions were run on a gel to detect cleavage. A control without gRNA was included. (B) Analysis of combinations of gRNAs and Cas9 sources for X-shredding. Average male frequencies in the F1 progeny are shown for each parental genotype with a single copy of βtub85Dtub85D-cas9 transgene (1X), two copies of βtub85Dtub85D-cas9 transgene (2X) or one copy of nos-cas9 (grey bars). All lines were crossed to wild type w individuals. The reciprocal cross (female ctrl) or heterozygote βtub85Dtub85D-cas9/ + or nos-cas9/ + without gRNA (no gRNA) were used as control. The black arrow indicates gRNAs in the multiplex array and the red dotted line indicates an unbiased sex-ratio. Crosses were set as pools of males and females or as multiple male single crosses in which case error bars indicate the mean ± SD for a minimum of ten independent single crosses. For all crosses n indicates the total number of individuals (males + females) in the F1 progeny counted. (C) Developmental survival analysis of the F1 progeny of Muc14a_6/βtub85Dtub85D-cas9 males crossed to w females compared to w and βtub85Dtub85D-cas9/ + control males crossed to w females. n indicates the number of individuals recorded at every developmental stage (males + females) in the F1 progeny. Bars indicate means ± SD for at least ten independent single crosses. Statistical significance was calculated with a t test assuming unequal variance. ** p

    Techniques Used: CRISPR, In Vitro, Binding Assay, Polymerase Chain Reaction, Amplification, Multiplex Assay

    4) Product Images from "Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis"

    Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky067

    Principles of Darwin Assembly. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the cut strand degraded by exonuclease III (1). Boundary and inner (mutagenic) oligonucleotides are annealed to the ssDNA plasmid (2). Key features of the oligonucleotides are highlighted: 5′-boundary oligonucleotide is 5′-biotinylated; non-complementary overhangs are shown in blue with Type IIs endonuclease recognition sites shown in white; mutations are shown as red X in the inner oligonucleotides; 3′-boundary oligonucleotide is protected at its 3′-end. After annealing, primers are extended and ligated in an isothermal assembly reaction (3). The assembled strand can be isolated by paramagnetic streptavidin-coated beads (4) and purified by alkali washing prior to PCR using outnested priming sites (5) and cloning (6) using the type IIS restriction sites (white dots). The purification step (4) is not always necessary but we found it improved PCR performance, especially for long assembly reactions ( > 1 kb).
    Figure Legend Snippet: Principles of Darwin Assembly. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the cut strand degraded by exonuclease III (1). Boundary and inner (mutagenic) oligonucleotides are annealed to the ssDNA plasmid (2). Key features of the oligonucleotides are highlighted: 5′-boundary oligonucleotide is 5′-biotinylated; non-complementary overhangs are shown in blue with Type IIs endonuclease recognition sites shown in white; mutations are shown as red X in the inner oligonucleotides; 3′-boundary oligonucleotide is protected at its 3′-end. After annealing, primers are extended and ligated in an isothermal assembly reaction (3). The assembled strand can be isolated by paramagnetic streptavidin-coated beads (4) and purified by alkali washing prior to PCR using outnested priming sites (5) and cloning (6) using the type IIS restriction sites (white dots). The purification step (4) is not always necessary but we found it improved PCR performance, especially for long assembly reactions ( > 1 kb).

    Techniques Used: Plasmid Preparation, Isolation, Purification, Polymerase Chain Reaction, Clone Assay

    Darwin Assembly using a θ oligonucleotide. Here, a single θ oligonucleotide is used in place of the two boundary oligonucleotides allowing enzymatic cleanup after the assembly reaction. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the nicked strand degraded by exonuclease III (1). Inner oligonucleotides and a single θ oligonucleotide are annealed to the ssDNA plasmid (2). The θ oligonucleotide encodes both assembly priming and termination sequences linked by a flexible linker such that successful assembly of the mutated strand results in a closed circle (3). The template plasmid can now be linearized (e.g. at the yellow dot, by adding a targeting oligonucleotide and appropriate restriction endonuclease) and both exonuclease I and exonuclease III added to degrade any non-circular DNA (4). The mutated gene can now be amplified from the closed circle by PCR (5) and cloned into a fresh vector (6) using the type IIS restriction sites (white dots).
    Figure Legend Snippet: Darwin Assembly using a θ oligonucleotide. Here, a single θ oligonucleotide is used in place of the two boundary oligonucleotides allowing enzymatic cleanup after the assembly reaction. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the nicked strand degraded by exonuclease III (1). Inner oligonucleotides and a single θ oligonucleotide are annealed to the ssDNA plasmid (2). The θ oligonucleotide encodes both assembly priming and termination sequences linked by a flexible linker such that successful assembly of the mutated strand results in a closed circle (3). The template plasmid can now be linearized (e.g. at the yellow dot, by adding a targeting oligonucleotide and appropriate restriction endonuclease) and both exonuclease I and exonuclease III added to degrade any non-circular DNA (4). The mutated gene can now be amplified from the closed circle by PCR (5) and cloned into a fresh vector (6) using the type IIS restriction sites (white dots).

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

    Darwin assembled TgoT DNA polymerase library. ( A ) Five separate sequencing reactions (range and reads shown in blue) were required to sample the diversity introduced across the eight target residues (shown in red along the TgoT gene). Mutations included focused degeneracies (e.g. YWC used against Y384) or ‘small intelligent’ (S-int) diversity (NDT, VMA, ATG and TGG oligonucleotides mixed in a 12:6:1:1 ratio). Resulting incorporation is shown in box plots with outliers explicitly labelled. Wild-type contamination was determined from positions where diversity excluded those sequences (N.A.: not applicable). As with the T7 RNA polymerase library, incorporation trends and biases were analysed to identify any biases in assembly. Ranked incorporation frequencies are shown for the residues targeted with ‘small intelligent’ diversity, and the top three highest (greens) and lowest (oranges) ranked codons (based on a straight sum of ranks) are highlighted ( B ). Outnest PCR of the TgoT DNA polymerase library (expected product of 2501 bp) showing that the final PCR can be carried out with either A- (MyTaq) or B-family (Q5) polymerases ( C ). MW: 1 kb ladder (NEB). NT: no template PCR control.
    Figure Legend Snippet: Darwin assembled TgoT DNA polymerase library. ( A ) Five separate sequencing reactions (range and reads shown in blue) were required to sample the diversity introduced across the eight target residues (shown in red along the TgoT gene). Mutations included focused degeneracies (e.g. YWC used against Y384) or ‘small intelligent’ (S-int) diversity (NDT, VMA, ATG and TGG oligonucleotides mixed in a 12:6:1:1 ratio). Resulting incorporation is shown in box plots with outliers explicitly labelled. Wild-type contamination was determined from positions where diversity excluded those sequences (N.A.: not applicable). As with the T7 RNA polymerase library, incorporation trends and biases were analysed to identify any biases in assembly. Ranked incorporation frequencies are shown for the residues targeted with ‘small intelligent’ diversity, and the top three highest (greens) and lowest (oranges) ranked codons (based on a straight sum of ranks) are highlighted ( B ). Outnest PCR of the TgoT DNA polymerase library (expected product of 2501 bp) showing that the final PCR can be carried out with either A- (MyTaq) or B-family (Q5) polymerases ( C ). MW: 1 kb ladder (NEB). NT: no template PCR control.

    Techniques Used: Sequencing, Polymerase Chain Reaction

    5) Product Images from "Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis"

    Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky067

    Principles of Darwin Assembly. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the cut strand degraded by exonuclease III (1). Boundary and inner (mutagenic) oligonucleotides are annealed to the ssDNA plasmid (2). Key features of the oligonucleotides are highlighted: 5′-boundary oligonucleotide is 5′-biotinylated; non-complementary overhangs are shown in blue with Type IIs endonuclease recognition sites shown in white; mutations are shown as red X in the inner oligonucleotides; 3′-boundary oligonucleotide is protected at its 3′-end. After annealing, primers are extended and ligated in an isothermal assembly reaction (3). The assembled strand can be isolated by paramagnetic streptavidin-coated beads (4) and purified by alkali washing prior to PCR using outnested priming sites (5) and cloning (6) using the type IIS restriction sites (white dots). The purification step (4) is not always necessary but we found it improved PCR performance, especially for long assembly reactions ( > 1 kb).
    Figure Legend Snippet: Principles of Darwin Assembly. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the cut strand degraded by exonuclease III (1). Boundary and inner (mutagenic) oligonucleotides are annealed to the ssDNA plasmid (2). Key features of the oligonucleotides are highlighted: 5′-boundary oligonucleotide is 5′-biotinylated; non-complementary overhangs are shown in blue with Type IIs endonuclease recognition sites shown in white; mutations are shown as red X in the inner oligonucleotides; 3′-boundary oligonucleotide is protected at its 3′-end. After annealing, primers are extended and ligated in an isothermal assembly reaction (3). The assembled strand can be isolated by paramagnetic streptavidin-coated beads (4) and purified by alkali washing prior to PCR using outnested priming sites (5) and cloning (6) using the type IIS restriction sites (white dots). The purification step (4) is not always necessary but we found it improved PCR performance, especially for long assembly reactions ( > 1 kb).

    Techniques Used: Plasmid Preparation, Isolation, Purification, Polymerase Chain Reaction, Clone Assay

    Darwin Assembly using a θ oligonucleotide. Here, a single θ oligonucleotide is used in place of the two boundary oligonucleotides allowing enzymatic cleanup after the assembly reaction. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the nicked strand degraded by exonuclease III (1). Inner oligonucleotides and a single θ oligonucleotide are annealed to the ssDNA plasmid (2). The θ oligonucleotide encodes both assembly priming and termination sequences linked by a flexible linker such that successful assembly of the mutated strand results in a closed circle (3). The template plasmid can now be linearized (e.g. at the yellow dot, by adding a targeting oligonucleotide and appropriate restriction endonuclease) and both exonuclease I and exonuclease III added to degrade any non-circular DNA (4). The mutated gene can now be amplified from the closed circle by PCR (5) and cloned into a fresh vector (6) using the type IIS restriction sites (white dots).
    Figure Legend Snippet: Darwin Assembly using a θ oligonucleotide. Here, a single θ oligonucleotide is used in place of the two boundary oligonucleotides allowing enzymatic cleanup after the assembly reaction. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the nicked strand degraded by exonuclease III (1). Inner oligonucleotides and a single θ oligonucleotide are annealed to the ssDNA plasmid (2). The θ oligonucleotide encodes both assembly priming and termination sequences linked by a flexible linker such that successful assembly of the mutated strand results in a closed circle (3). The template plasmid can now be linearized (e.g. at the yellow dot, by adding a targeting oligonucleotide and appropriate restriction endonuclease) and both exonuclease I and exonuclease III added to degrade any non-circular DNA (4). The mutated gene can now be amplified from the closed circle by PCR (5) and cloned into a fresh vector (6) using the type IIS restriction sites (white dots).

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

    Darwin assembled TgoT DNA polymerase library. ( A ) Five separate sequencing reactions (range and reads shown in blue) were required to sample the diversity introduced across the eight target residues (shown in red along the TgoT gene). Mutations included focused degeneracies (e.g. YWC used against Y384) or ‘small intelligent’ (S-int) diversity (NDT, VMA, ATG and TGG oligonucleotides mixed in a 12:6:1:1 ratio). Resulting incorporation is shown in box plots with outliers explicitly labelled. Wild-type contamination was determined from positions where diversity excluded those sequences (N.A.: not applicable). As with the T7 RNA polymerase library, incorporation trends and biases were analysed to identify any biases in assembly. Ranked incorporation frequencies are shown for the residues targeted with ‘small intelligent’ diversity, and the top three highest (greens) and lowest (oranges) ranked codons (based on a straight sum of ranks) are highlighted ( B ). Outnest PCR of the TgoT DNA polymerase library (expected product of 2501 bp) showing that the final PCR can be carried out with either A- (MyTaq) or B-family (Q5) polymerases ( C ). MW: 1 kb ladder (NEB). NT: no template PCR control.
    Figure Legend Snippet: Darwin assembled TgoT DNA polymerase library. ( A ) Five separate sequencing reactions (range and reads shown in blue) were required to sample the diversity introduced across the eight target residues (shown in red along the TgoT gene). Mutations included focused degeneracies (e.g. YWC used against Y384) or ‘small intelligent’ (S-int) diversity (NDT, VMA, ATG and TGG oligonucleotides mixed in a 12:6:1:1 ratio). Resulting incorporation is shown in box plots with outliers explicitly labelled. Wild-type contamination was determined from positions where diversity excluded those sequences (N.A.: not applicable). As with the T7 RNA polymerase library, incorporation trends and biases were analysed to identify any biases in assembly. Ranked incorporation frequencies are shown for the residues targeted with ‘small intelligent’ diversity, and the top three highest (greens) and lowest (oranges) ranked codons (based on a straight sum of ranks) are highlighted ( B ). Outnest PCR of the TgoT DNA polymerase library (expected product of 2501 bp) showing that the final PCR can be carried out with either A- (MyTaq) or B-family (Q5) polymerases ( C ). MW: 1 kb ladder (NEB). NT: no template PCR control.

    Techniques Used: Sequencing, Polymerase Chain Reaction

    6) Product Images from "Identification of the First Gene Transfer Agent (GTA) Small Terminase in Rhodobacter capsulatus and Its Role in GTA Production and Packaging of DNA"

    Article Title: Identification of the First Gene Transfer Agent (GTA) Small Terminase in Rhodobacter capsulatus and Its Role in GTA Production and Packaging of DNA

    Journal: Journal of Virology

    doi: 10.1128/JVI.01328-19

    RcGTA gp1 in vitro DNA binding. (A) Representative agarose gel (0.8%, wt/vol) showing the stated concentrations of gp1 protein binding to DNA in an electrophoretic mobility shift assay (EMSA). The locations of unbound and shifted DNA are annotated. Substrate DNA in the assay shown is a 1.4-kbp PCR amplification of an arbitrarily chosen region flanking the rcc01398 gene from R. capsulatus (amplified using rcc01398 forward and reverse primers [ Table 3 ]). Bioline HyperLadder 1kb DNA marker is shown for size comparison (lane M). (B) Quantification of EMSAs by band intensity analysis. Data shown are the average results of two EMSAs carried out independently in time and with different DNA substrates (flanking the rcc01397 and rcc01398 genes). Individual data points are plotted as well as the mean line.
    Figure Legend Snippet: RcGTA gp1 in vitro DNA binding. (A) Representative agarose gel (0.8%, wt/vol) showing the stated concentrations of gp1 protein binding to DNA in an electrophoretic mobility shift assay (EMSA). The locations of unbound and shifted DNA are annotated. Substrate DNA in the assay shown is a 1.4-kbp PCR amplification of an arbitrarily chosen region flanking the rcc01398 gene from R. capsulatus (amplified using rcc01398 forward and reverse primers [ Table 3 ]). Bioline HyperLadder 1kb DNA marker is shown for size comparison (lane M). (B) Quantification of EMSAs by band intensity analysis. Data shown are the average results of two EMSAs carried out independently in time and with different DNA substrates (flanking the rcc01397 and rcc01398 genes). Individual data points are plotted as well as the mean line.

    Techniques Used: In Vitro, Binding Assay, Agarose Gel Electrophoresis, Protein Binding, Electrophoretic Mobility Shift Assay, Polymerase Chain Reaction, Amplification, Marker

    7) Product Images from "DNA polymerase stalling at structured DNA constrains the expansion of Short Tandem Repeats"

    Article Title: DNA polymerase stalling at structured DNA constrains the expansion of Short Tandem Repeats

    Journal: bioRxiv

    doi: 10.1101/2020.06.20.162743

    Pooled measurement of DNA polymerase stalling at STRs. (a) Overview of the high-throughput primer extension assay used to monitor DNA synthesis at designed sequences. A library of 20,000 sequences comprising all STR permutations at three different lengths together with control structured DNA sequences was synthesised on a programmable microarray, eluted and inserted into a phagemid vector. After PCR amplification, insertion into a phagemid vector and bacterial amplification, circular single-stranded DNA templates were produced using a M13KO7 helper phage. Fluorescently labelled primer (P3) and structures annealing were performed before initiating DNA synthesis through the addition of T7 DNA polymerase. Primers are then either fully extended to the length of the circular template or the extension is stopped within STRs if the DNA polymerases stall at structured DNAs. (b) Extended and stalled products were then analysed by denaturing Poly Acrylamide Gel (PAGE) electrophoresis, recovered from the gel matrix and prepared for high throughput sequencing. DNA polymerase stalling was then quantified by analysing the enrichment of each sequence form the library in the stalled and extended fractions. Representative fluorescence gel imaging of primer extension reactions on templates containing a G-quadruplex (G4) structure, a mutated G4 or the entire DNA library, stopped after the indicated times, are reported for comparison. Blue and red arrows indicate the position of the extended and stalled products respectively. The green line highlights the presence of transient stall sites that disappear overtime.
    Figure Legend Snippet: Pooled measurement of DNA polymerase stalling at STRs. (a) Overview of the high-throughput primer extension assay used to monitor DNA synthesis at designed sequences. A library of 20,000 sequences comprising all STR permutations at three different lengths together with control structured DNA sequences was synthesised on a programmable microarray, eluted and inserted into a phagemid vector. After PCR amplification, insertion into a phagemid vector and bacterial amplification, circular single-stranded DNA templates were produced using a M13KO7 helper phage. Fluorescently labelled primer (P3) and structures annealing were performed before initiating DNA synthesis through the addition of T7 DNA polymerase. Primers are then either fully extended to the length of the circular template or the extension is stopped within STRs if the DNA polymerases stall at structured DNAs. (b) Extended and stalled products were then analysed by denaturing Poly Acrylamide Gel (PAGE) electrophoresis, recovered from the gel matrix and prepared for high throughput sequencing. DNA polymerase stalling was then quantified by analysing the enrichment of each sequence form the library in the stalled and extended fractions. Representative fluorescence gel imaging of primer extension reactions on templates containing a G-quadruplex (G4) structure, a mutated G4 or the entire DNA library, stopped after the indicated times, are reported for comparison. Blue and red arrows indicate the position of the extended and stalled products respectively. The green line highlights the presence of transient stall sites that disappear overtime.

    Techniques Used: High Throughput Screening Assay, Primer Extension Assay, DNA Synthesis, Microarray, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Produced, Acrylamide Gel Assay, Polyacrylamide Gel Electrophoresis, Electrophoresis, Next-Generation Sequencing, Sequencing, Fluorescence, Imaging

    8) Product Images from "DNA polymerase stalling at structured DNA constrains the expansion of Short Tandem Repeats"

    Article Title: DNA polymerase stalling at structured DNA constrains the expansion of Short Tandem Repeats

    Journal: bioRxiv

    doi: 10.1101/2020.06.20.162743

    Pooled measurement of DNA polymerase stalling at STRs. (a) Overview of the high-throughput primer extension assay used to monitor DNA synthesis at designed sequences. A library of 20,000 sequences comprising all STR permutations at three different lengths together with control structured DNA sequences was synthesised on a programmable microarray, eluted and inserted into a phagemid vector. After PCR amplification, insertion into a phagemid vector and bacterial amplification, circular single-stranded DNA templates were produced using a M13KO7 helper phage. Fluorescently labelled primer (P3) and structures annealing were performed before initiating DNA synthesis through the addition of T7 DNA polymerase. Primers are then either fully extended to the length of the circular template or the extension is stopped within STRs if the DNA polymerases stall at structured DNAs. (b) Extended and stalled products were then analysed by denaturing Poly Acrylamide Gel (PAGE) electrophoresis, recovered from the gel matrix and prepared for high throughput sequencing. DNA polymerase stalling was then quantified by analysing the enrichment of each sequence form the library in the stalled and extended fractions. Representative fluorescence gel imaging of primer extension reactions on templates containing a G-quadruplex (G4) structure, a mutated G4 or the entire DNA library, stopped after the indicated times, are reported for comparison. Blue and red arrows indicate the position of the extended and stalled products respectively. The green line highlights the presence of transient stall sites that disappear overtime.
    Figure Legend Snippet: Pooled measurement of DNA polymerase stalling at STRs. (a) Overview of the high-throughput primer extension assay used to monitor DNA synthesis at designed sequences. A library of 20,000 sequences comprising all STR permutations at three different lengths together with control structured DNA sequences was synthesised on a programmable microarray, eluted and inserted into a phagemid vector. After PCR amplification, insertion into a phagemid vector and bacterial amplification, circular single-stranded DNA templates were produced using a M13KO7 helper phage. Fluorescently labelled primer (P3) and structures annealing were performed before initiating DNA synthesis through the addition of T7 DNA polymerase. Primers are then either fully extended to the length of the circular template or the extension is stopped within STRs if the DNA polymerases stall at structured DNAs. (b) Extended and stalled products were then analysed by denaturing Poly Acrylamide Gel (PAGE) electrophoresis, recovered from the gel matrix and prepared for high throughput sequencing. DNA polymerase stalling was then quantified by analysing the enrichment of each sequence form the library in the stalled and extended fractions. Representative fluorescence gel imaging of primer extension reactions on templates containing a G-quadruplex (G4) structure, a mutated G4 or the entire DNA library, stopped after the indicated times, are reported for comparison. Blue and red arrows indicate the position of the extended and stalled products respectively. The green line highlights the presence of transient stall sites that disappear overtime.

    Techniques Used: High Throughput Screening Assay, Primer Extension Assay, DNA Synthesis, Microarray, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Produced, Acrylamide Gel Assay, Polyacrylamide Gel Electrophoresis, Electrophoresis, Next-Generation Sequencing, Sequencing, Fluorescence, Imaging

    9) Product Images from "DNA polymerase stalling at structured DNA constrains the expansion of short tandem repeats"

    Article Title: DNA polymerase stalling at structured DNA constrains the expansion of short tandem repeats

    Journal: Genome Biology

    doi: 10.1186/s13059-020-02124-x

    Pooled measurement of DNA polymerase stalling at STRs. a Overview of the high-throughput primer extension assay used to monitor DNA synthesis at designed sequences. A library of 20,000 sequences comprising all STR permutations at three different lengths together with control structured DNA sequences was synthesised on a programmable microarray, eluted and inserted into a phagemid vector. After PCR amplification, insertion into a phagemid vector and bacterial amplification, circular single-stranded DNA templates were produced using a M13KO7 helper phage. Fluorescently labelled primer (P3) and structure annealing were performed before initiating DNA synthesis through the addition of T7 DNA polymerase. Primers are then either fully extended to the length of the circular template, or the extension is stopped within STRs if the DNA polymerases stall at structured DNAs. b Extended and stalled products were then analysed by denaturing poly acrylamide gel electrophoresis (PAGE), recovered from the gel matrix and prepared for high-throughput sequencing. DNA polymerase stalling was then quantified by analysing the enrichment of each sequence from the library in the stalled and extended fractions. Representative fluorescence gel imaging of primer extension reactions on templates containing a G-quadruplex (G4) structure, a mutated G4 or the entire DNA library, stopped after the indicated times, is reported for comparison. Blue and red arrows indicate the position of the extended and stalled products respectively. The green line highlights the presence of transient stall sites that disappear over time
    Figure Legend Snippet: Pooled measurement of DNA polymerase stalling at STRs. a Overview of the high-throughput primer extension assay used to monitor DNA synthesis at designed sequences. A library of 20,000 sequences comprising all STR permutations at three different lengths together with control structured DNA sequences was synthesised on a programmable microarray, eluted and inserted into a phagemid vector. After PCR amplification, insertion into a phagemid vector and bacterial amplification, circular single-stranded DNA templates were produced using a M13KO7 helper phage. Fluorescently labelled primer (P3) and structure annealing were performed before initiating DNA synthesis through the addition of T7 DNA polymerase. Primers are then either fully extended to the length of the circular template, or the extension is stopped within STRs if the DNA polymerases stall at structured DNAs. b Extended and stalled products were then analysed by denaturing poly acrylamide gel electrophoresis (PAGE), recovered from the gel matrix and prepared for high-throughput sequencing. DNA polymerase stalling was then quantified by analysing the enrichment of each sequence from the library in the stalled and extended fractions. Representative fluorescence gel imaging of primer extension reactions on templates containing a G-quadruplex (G4) structure, a mutated G4 or the entire DNA library, stopped after the indicated times, is reported for comparison. Blue and red arrows indicate the position of the extended and stalled products respectively. The green line highlights the presence of transient stall sites that disappear over time

    Techniques Used: High Throughput Screening Assay, Primer Extension Assay, DNA Synthesis, Microarray, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Produced, Polyacrylamide Gel Electrophoresis, Next-Generation Sequencing, Sequencing, Fluorescence, Imaging

    10) Product Images from "Single telomere length analysis in Ustilago maydis, a high-resolution tool for examining fungal telomere length distribution and C-strand 5’-end processing"

    Article Title: Single telomere length analysis in Ustilago maydis, a high-resolution tool for examining fungal telomere length distribution and C-strand 5’-end processing

    Journal: Microbial Cell

    doi: 10.15698/mic2018.09.645

    FIGURE 1: STELA protocol and investigation of UT4/5-containing telomeres. (A) Schematic illustration of the structure of UT4 and UT5-containing telomeres in U. maydis . The use of telorette oligos to modify the C-strand and the use of primers (UT4/5-F and teltail) to generate STELA products are also illustrated. (B) Four individual STELA PCR reactions for UT4/5 telomeres were performed using 2.5 pg of ligated wild type DNA as the template and shown on the left. A parallel Southern analysis is shown on the right. The same UT4/5 subtelomeric probe was used to detect telomere fragments in both analyses. (C) STELA assays were performed using 5 pg wild type DNA as the template, and the UT4/5-F and teltail oligos as primers. Following gel electrophoresis and transfer to a nylon membrane, the products were first detected using a UT4/5 subtelomeric probe (left panel). Subsequently, the UT4/5 probe was stripped from the membrane and the products re-analyzed using a TTAGGG repeat probe (middle panel). The sizes of the STELA fragments in the middle panel were determined using TESLA software. The lengths of the telomere tracts were then calculated by subtracting the subtelomere length (~630 bp), and then plotted (right). Error bars designate standard error of means.
    Figure Legend Snippet: FIGURE 1: STELA protocol and investigation of UT4/5-containing telomeres. (A) Schematic illustration of the structure of UT4 and UT5-containing telomeres in U. maydis . The use of telorette oligos to modify the C-strand and the use of primers (UT4/5-F and teltail) to generate STELA products are also illustrated. (B) Four individual STELA PCR reactions for UT4/5 telomeres were performed using 2.5 pg of ligated wild type DNA as the template and shown on the left. A parallel Southern analysis is shown on the right. The same UT4/5 subtelomeric probe was used to detect telomere fragments in both analyses. (C) STELA assays were performed using 5 pg wild type DNA as the template, and the UT4/5-F and teltail oligos as primers. Following gel electrophoresis and transfer to a nylon membrane, the products were first detected using a UT4/5 subtelomeric probe (left panel). Subsequently, the UT4/5 probe was stripped from the membrane and the products re-analyzed using a TTAGGG repeat probe (middle panel). The sizes of the STELA fragments in the middle panel were determined using TESLA software. The lengths of the telomere tracts were then calculated by subtracting the subtelomere length (~630 bp), and then plotted (right). Error bars designate standard error of means.

    Techniques Used: Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Software

    11) Product Images from "Engineering Maize rayado fino virus for virus‐induced gene silencing. Engineering Maize rayado fino virus for virus‐induced gene silencing"

    Article Title: Engineering Maize rayado fino virus for virus‐induced gene silencing. Engineering Maize rayado fino virus for virus‐induced gene silencing

    Journal: Plant Direct

    doi: 10.1002/pld3.224

    Utility of the MRFV VIGS system on different maize inbred lines. (a) MRFV symptoms and PDS photobleaching induced by MRFV‐PDS 120 on B73, Mo17, and Va35 maize inbred lines compared to plants inoculated with MRFV‐WT or noninoculated healthy controls (HC). (b) RT‐PCR detection of MRFV‐PDS 120 and MRFV‐WT in systemic leaves of B73, Mo17, and Va35 plants 30 dpi. Noninoculated plants (HC) and water (H 2 O) were included as negative controls; MRFV‐PDS 120 plasmid (PL) served as a PCR‐positive control (not shown). M: 100 bp DNA marker. (c) Northern blot analysis of VIGS induced by MRFV‐PDS 120 on B73, Mo17, and Va35 maize inbred lines. Blot hybridizations were as described for Figure 3 . PDS mRNA and siRNA levels in MRFV‐PDS 120 ‐infected plants are shown (lanes 1–2; 7–8; and 13–14) compared to MRFV infected (lanes 3–4; 9–10; and 15–16) and healthy plants (lanes 5–6; 11–12; and 17–18). The relative levels of PDS mRNA were determined as described in Figure 3
    Figure Legend Snippet: Utility of the MRFV VIGS system on different maize inbred lines. (a) MRFV symptoms and PDS photobleaching induced by MRFV‐PDS 120 on B73, Mo17, and Va35 maize inbred lines compared to plants inoculated with MRFV‐WT or noninoculated healthy controls (HC). (b) RT‐PCR detection of MRFV‐PDS 120 and MRFV‐WT in systemic leaves of B73, Mo17, and Va35 plants 30 dpi. Noninoculated plants (HC) and water (H 2 O) were included as negative controls; MRFV‐PDS 120 plasmid (PL) served as a PCR‐positive control (not shown). M: 100 bp DNA marker. (c) Northern blot analysis of VIGS induced by MRFV‐PDS 120 on B73, Mo17, and Va35 maize inbred lines. Blot hybridizations were as described for Figure 3 . PDS mRNA and siRNA levels in MRFV‐PDS 120 ‐infected plants are shown (lanes 1–2; 7–8; and 13–14) compared to MRFV infected (lanes 3–4; 9–10; and 15–16) and healthy plants (lanes 5–6; 11–12; and 17–18). The relative levels of PDS mRNA were determined as described in Figure 3

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Polymerase Chain Reaction, Positive Control, Marker, Northern Blot, Infection

    (a) Transmissibility of MRFV‐PDS 120 by D. maidis ( Dm ) and RT‐PCR analysis (primers sm151 and 152) of insert stability. Replicated experiments showing virus symptoms and chlorophyll photobleaching phenotype induced by D. maidis‐ transmitted MRFV‐PDS 120 at 30 dpi compared to MRFV‐WT and noninoculated plants (i, iii and v); and corresponding RT‐PCR assays of MRFV‐PDS 120 and MRFV‐WT in systemic leaves of infected plants (ii, iv and vi). Healthy plants (HC) and water (H 2 O) were included as negative controls, and the MRFV‐PDS 120 plasmid (PL) as a positive control. M: 100 bp DNA marker. (b) Stability of MRFV‐PDS 120 through D. maidis ( Dm ) passaging. (i) Virus symptoms and chlorophyll photo bleaching phenotype induced by MRFV‐PDS 120 at 30 dpi after passage 2 acquisition and transmission by D. maidis , compared to MRFV‐WT and noninoculated plants, and (ii) corresponding RT‐PCR assays of MRFV‐PDS 120 and MRFV‐WT in systemic leaves of infected plants . The controls and DNA marker were included as described above. (iii) Virus symptoms and chlorophyll photobleaching phenotype induced by MRFV‐PDS 120 at 30 dpi after passage three acquisition and transmission by D. maidis , compared to MRFV‐WT and noninoculated plants, and (iv) corresponding RT‐PCR assays of MRFV‐PDS 120 and MRFV‐WT in systemic leaves of infected plants . The controls were included as described above
    Figure Legend Snippet: (a) Transmissibility of MRFV‐PDS 120 by D. maidis ( Dm ) and RT‐PCR analysis (primers sm151 and 152) of insert stability. Replicated experiments showing virus symptoms and chlorophyll photobleaching phenotype induced by D. maidis‐ transmitted MRFV‐PDS 120 at 30 dpi compared to MRFV‐WT and noninoculated plants (i, iii and v); and corresponding RT‐PCR assays of MRFV‐PDS 120 and MRFV‐WT in systemic leaves of infected plants (ii, iv and vi). Healthy plants (HC) and water (H 2 O) were included as negative controls, and the MRFV‐PDS 120 plasmid (PL) as a positive control. M: 100 bp DNA marker. (b) Stability of MRFV‐PDS 120 through D. maidis ( Dm ) passaging. (i) Virus symptoms and chlorophyll photo bleaching phenotype induced by MRFV‐PDS 120 at 30 dpi after passage 2 acquisition and transmission by D. maidis , compared to MRFV‐WT and noninoculated plants, and (ii) corresponding RT‐PCR assays of MRFV‐PDS 120 and MRFV‐WT in systemic leaves of infected plants . The controls and DNA marker were included as described above. (iii) Virus symptoms and chlorophyll photobleaching phenotype induced by MRFV‐PDS 120 at 30 dpi after passage three acquisition and transmission by D. maidis , compared to MRFV‐WT and noninoculated plants, and (iv) corresponding RT‐PCR assays of MRFV‐PDS 120 and MRFV‐WT in systemic leaves of infected plants . The controls were included as described above

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Infection, Plasmid Preparation, Positive Control, Marker, Passaging, Transmission Assay

    Testing of the stability of MRFV‐PDS 120 using serial passage inoculations with crude plant sap. (a) MRFV‐PDS 120 symptoms and chlorophyll photobleaching phenotype at 30 dpi for each passage, compared to MRFV‐WT and noninoculated plants (HC). (b) RT‐PCR analysis (primers sm151 and sm152) of the integrity of MRFV‐PDS 120 at each of the four passages. All the MRFV‐PDS 120 ‐infected plants obtained for each passage were RT‐PCR assayed both at 15 dpi (not shown) and at 60 dpi (representative gel shown here). Healthy plants (HC) and water (H 2 O) were included as negative controls, and MRFV‐PDS 120 plasmid (PL) as a positive control. M: 100 bp DNA marker.
    Figure Legend Snippet: Testing of the stability of MRFV‐PDS 120 using serial passage inoculations with crude plant sap. (a) MRFV‐PDS 120 symptoms and chlorophyll photobleaching phenotype at 30 dpi for each passage, compared to MRFV‐WT and noninoculated plants (HC). (b) RT‐PCR analysis (primers sm151 and sm152) of the integrity of MRFV‐PDS 120 at each of the four passages. All the MRFV‐PDS 120 ‐infected plants obtained for each passage were RT‐PCR assayed both at 15 dpi (not shown) and at 60 dpi (representative gel shown here). Healthy plants (HC) and water (H 2 O) were included as negative controls, and MRFV‐PDS 120 plasmid (PL) as a positive control. M: 100 bp DNA marker.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Infection, Plasmid Preparation, Positive Control, Marker

    Viability of MRFV HEL/POL junction as insertion site. (a) Location of primers (sm151 and sm152) used for RT‐PCR detection of MRFV‐PDS 120 and MRFV‐WT. The primers straddle the HEL/POL junction and amplify 1,164 bp in MRFV‐PDS 120 and 963bp in wild‐type MRFV. (b) Virus symptoms and the chlorophyll photobleaching phenotype induced by MRFV‐PDS 120 at 30 dpi, compared to leaves of plants inoculated with MRFV without insert and noninoculated leaves (HC). (c) RT‐PCR analysis of virus accumulation in systemic leaves at 30 days post‐inoculation. The three gels show RT‐PCR detection of MRFV‐PDS 120 and MRFV‐WT in inoculated plants in three replicated experiments, with RNA from noninoculated plants (HC) and water (H 2 O) included as negative control templates; and the MRFV‐PDS 120 plasmid (PL) serving as a positive control. Blank lanes in MRFV‐PDS 120 inoculations represent nonsymptomatic plants. M: 100 bp DNA marker. (d) RT‐PCR analysis of insert retention in MRFV‐PDS 120 at 60 days post‐inoculation. RT‐PCR was repeated at 60 dpi only for RT‐PCR positive (symptomatic/successfully inoculated) MRFV‐PDS 120 plants in (c) above, with controls and DNA marker included as described above. This was done for all the three replicated experiments. (e) RT‐PCR assays for virus accumulation in tassels and silks for a subset of MRFV‐PDS 120 symptomatic plants. Controls and DNA marker were included as described above. (f) Transmission electron micrographs of MRFV‐PDS 120 (left panel) and MRFV‐WT particles (right panel) in maize (Silver Queen) extract
    Figure Legend Snippet: Viability of MRFV HEL/POL junction as insertion site. (a) Location of primers (sm151 and sm152) used for RT‐PCR detection of MRFV‐PDS 120 and MRFV‐WT. The primers straddle the HEL/POL junction and amplify 1,164 bp in MRFV‐PDS 120 and 963bp in wild‐type MRFV. (b) Virus symptoms and the chlorophyll photobleaching phenotype induced by MRFV‐PDS 120 at 30 dpi, compared to leaves of plants inoculated with MRFV without insert and noninoculated leaves (HC). (c) RT‐PCR analysis of virus accumulation in systemic leaves at 30 days post‐inoculation. The three gels show RT‐PCR detection of MRFV‐PDS 120 and MRFV‐WT in inoculated plants in three replicated experiments, with RNA from noninoculated plants (HC) and water (H 2 O) included as negative control templates; and the MRFV‐PDS 120 plasmid (PL) serving as a positive control. Blank lanes in MRFV‐PDS 120 inoculations represent nonsymptomatic plants. M: 100 bp DNA marker. (d) RT‐PCR analysis of insert retention in MRFV‐PDS 120 at 60 days post‐inoculation. RT‐PCR was repeated at 60 dpi only for RT‐PCR positive (symptomatic/successfully inoculated) MRFV‐PDS 120 plants in (c) above, with controls and DNA marker included as described above. This was done for all the three replicated experiments. (e) RT‐PCR assays for virus accumulation in tassels and silks for a subset of MRFV‐PDS 120 symptomatic plants. Controls and DNA marker were included as described above. (f) Transmission electron micrographs of MRFV‐PDS 120 (left panel) and MRFV‐WT particles (right panel) in maize (Silver Queen) extract

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Negative Control, Plasmid Preparation, Positive Control, Marker, Transmission Assay

    Capacity of HEL/POL junction to hold larger inserts. (a) Similar to MRFV‐PDS 120, molecular assays for MRFV‐PDS 231 used primers sm151 and 152 for RT‐PCR detection, amplifying 1,275 bp in MRFV‐PDS 231 and 963bp in MRFV‐WT. (b) Virus symptoms and chlorophyll photobleaching phenotype induced by MRFV‐PDS 231 at 30 dpi, compared to MRFV‐WT and noninoculated plants. (c) RT‐PCR analysis of virus accumulation in systemic leaves at 30 dpi. The gel shows RT‐PCR detection of MRFV‐PDS 231 and MRFV‐WT in systemic leaves of inoculated plants. Healthy plants (HC) and water (H 2 O) were included as negative controls and the MRFV‐PDS 231 plasmid (PL) as a positive control. M: 100 bp DNA marker. (d) Northern blot analysis of photobleaching phenotype induced by MRFV‐PDS 231 was as described in Figure 3 . Levels of PDS mRNA and siRNAs from MRFV‐PDS 231 ‐infected leaves is shown (lanes 1–4) compared to MRFV‐infected (lanes 5–6) and healthy plants (lanes 7–8). The relative levels of PDS mRNA were determined as described in Figure 3
    Figure Legend Snippet: Capacity of HEL/POL junction to hold larger inserts. (a) Similar to MRFV‐PDS 120, molecular assays for MRFV‐PDS 231 used primers sm151 and 152 for RT‐PCR detection, amplifying 1,275 bp in MRFV‐PDS 231 and 963bp in MRFV‐WT. (b) Virus symptoms and chlorophyll photobleaching phenotype induced by MRFV‐PDS 231 at 30 dpi, compared to MRFV‐WT and noninoculated plants. (c) RT‐PCR analysis of virus accumulation in systemic leaves at 30 dpi. The gel shows RT‐PCR detection of MRFV‐PDS 231 and MRFV‐WT in systemic leaves of inoculated plants. Healthy plants (HC) and water (H 2 O) were included as negative controls and the MRFV‐PDS 231 plasmid (PL) as a positive control. M: 100 bp DNA marker. (d) Northern blot analysis of photobleaching phenotype induced by MRFV‐PDS 231 was as described in Figure 3 . Levels of PDS mRNA and siRNAs from MRFV‐PDS 231 ‐infected leaves is shown (lanes 1–4) compared to MRFV‐infected (lanes 5–6) and healthy plants (lanes 7–8). The relative levels of PDS mRNA were determined as described in Figure 3

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Positive Control, Marker, Northern Blot, Infection

    Efficacy of MRFV VIGS system in silencing ZmlspH gene. (a) Molecular assays for MRFV‐LSP 210 used primers sm151 and 152 for RT‐PCR detection, amplifying 1,254 bp in MRFV‐LSP 210 and 963bp in MRFV‐WT. (b) MRFV symptoms and yellow (albino) VIGS phenotype induced by MRFV‐LSP 210 at 30 dpi, compared to plants inoculated with MRFV‐WT or noninoculated (HC). (c) RT‐PCR analysis of MRFV‐LSP 210 and MRFV‐WT accumulation in systemic leaves 30 dpi. Noninoculated plants (HC) were included as negative controls; MRFV‐LSP 210 plasmid (PL) served as a PCR‐positive control. M: 100 bp DNA marker. (d) Northern blot analysis of VIGS induced by MRFV‐LSP 210. RNA blots were hybridized with LSP‐specific RNA probes to detect LSP mRNAs or antisense siRNAs. rRNA: EtBr‐stained ribosomal RNA used as loading control. For small RNA gel, the major low molecular weight RNA species was EtBr‐stained for loading control prior to bloating of gel onto membrane. Levels of LSP mRNA and siRNAs from MRFV‐LSP 210 ‐infected plants (lanes 1–4) compared to MRFV‐infected (lanes 5–6) and healthy plants (lanes 7–8) are shown, with positions of 21‐ to 24‐nucleotide RNA size markers indicated by arrow heads. The relative levels of LSP mRNA were determined as described for PDS in Figure 3
    Figure Legend Snippet: Efficacy of MRFV VIGS system in silencing ZmlspH gene. (a) Molecular assays for MRFV‐LSP 210 used primers sm151 and 152 for RT‐PCR detection, amplifying 1,254 bp in MRFV‐LSP 210 and 963bp in MRFV‐WT. (b) MRFV symptoms and yellow (albino) VIGS phenotype induced by MRFV‐LSP 210 at 30 dpi, compared to plants inoculated with MRFV‐WT or noninoculated (HC). (c) RT‐PCR analysis of MRFV‐LSP 210 and MRFV‐WT accumulation in systemic leaves 30 dpi. Noninoculated plants (HC) were included as negative controls; MRFV‐LSP 210 plasmid (PL) served as a PCR‐positive control. M: 100 bp DNA marker. (d) Northern blot analysis of VIGS induced by MRFV‐LSP 210. RNA blots were hybridized with LSP‐specific RNA probes to detect LSP mRNAs or antisense siRNAs. rRNA: EtBr‐stained ribosomal RNA used as loading control. For small RNA gel, the major low molecular weight RNA species was EtBr‐stained for loading control prior to bloating of gel onto membrane. Levels of LSP mRNA and siRNAs from MRFV‐LSP 210 ‐infected plants (lanes 1–4) compared to MRFV‐infected (lanes 5–6) and healthy plants (lanes 7–8) are shown, with positions of 21‐ to 24‐nucleotide RNA size markers indicated by arrow heads. The relative levels of LSP mRNA were determined as described for PDS in Figure 3

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Polymerase Chain Reaction, Positive Control, Marker, Northern Blot, Staining, Molecular Weight, Infection

    12) Product Images from "Selection of an Efficient AAV Vector for Robust CNS Transgene Expression"

    Article Title: Selection of an Efficient AAV Vector for Robust CNS Transgene Expression

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1016/j.omtm.2019.10.007

    iTransduce Library for Selection of Novel AAV Capsids Capable of Efficient Transgene Expression in Target Tissue (A) Two-component system of the library construct. (1) Cre recombinase is driven by a minimal chicken β-actin (CBA) promoter. (2) p41 promoter-driven AAV9 capsid with random heptamer peptide is inserted between amino acids 588 and 589, cloned downstream of the Cre cassette. (B) Selection strategy. (Bi) The iTransduce library comprised of different peptide inserts expressed on the capsid (represented by different colors) is injected intravenously (i.v.) into an Ai9 transgenic mouse with a loxP-flanked STOP cassette upsteam of the tdTomato reporter gene, inserted into the Gt(ROSA)26Sor locus. AAV capsids able to enter the cell of interest but that do not functionally transduce the cell (no Cre expression) do not turn on tdTomato expression. Capsids that can mediate functional transduction (express Cre) will turn on tdTomato expression. (Bii) Cells are isolated from the organ of interest (e.g., brain), and transduced cells are sorted for tdTomato expression and optionally cell markers. (Biii) Capsid DNA is PCR amplified from the sorted cells, cloned back to the library vector, and repackaged for another round of selection. DNA sequencing analysis is utilized after each round to monitor the selection process.
    Figure Legend Snippet: iTransduce Library for Selection of Novel AAV Capsids Capable of Efficient Transgene Expression in Target Tissue (A) Two-component system of the library construct. (1) Cre recombinase is driven by a minimal chicken β-actin (CBA) promoter. (2) p41 promoter-driven AAV9 capsid with random heptamer peptide is inserted between amino acids 588 and 589, cloned downstream of the Cre cassette. (B) Selection strategy. (Bi) The iTransduce library comprised of different peptide inserts expressed on the capsid (represented by different colors) is injected intravenously (i.v.) into an Ai9 transgenic mouse with a loxP-flanked STOP cassette upsteam of the tdTomato reporter gene, inserted into the Gt(ROSA)26Sor locus. AAV capsids able to enter the cell of interest but that do not functionally transduce the cell (no Cre expression) do not turn on tdTomato expression. Capsids that can mediate functional transduction (express Cre) will turn on tdTomato expression. (Bii) Cells are isolated from the organ of interest (e.g., brain), and transduced cells are sorted for tdTomato expression and optionally cell markers. (Biii) Capsid DNA is PCR amplified from the sorted cells, cloned back to the library vector, and repackaged for another round of selection. DNA sequencing analysis is utilized after each round to monitor the selection process.

    Techniques Used: Selection, Expressing, Construct, Crocin Bleaching Assay, Clone Assay, Injection, Transgenic Assay, Functional Assay, Transduction, Isolation, Polymerase Chain Reaction, Amplification, Plasmid Preparation, DNA Sequencing

    13) Product Images from "Characterisation of resistance mechanisms developed by basal cell carcinoma cells in response to repeated cycles of Photodynamic Therapy"

    Article Title: Characterisation of resistance mechanisms developed by basal cell carcinoma cells in response to repeated cycles of Photodynamic Therapy

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-41313-y

    Localisation and expression of E-cadherin. ( A ) Immunofluorescence images of E-cadherin expression in green and DNA staining (DAPI) in blue. A secondary antibody control (AlexaFluor 488) for P cells is included. In CSZ, polygonal population is indicated with an asterisk and spindled cells with and an arrow. Scale bar: 40 µm. ( B ) A representative experiment of E-cadherin expression by Western Blot and densitometry graphics corresponding to three independent experiments are showed (Mean ± SD). Load control: β-actin. Separate gels where used for cell line and tumour cells. ROD (Relative optic density). ( C ) mRNA levels resulting from RT-PCR analysis. Relative data to their corresponding P or P T population are presented in the graph. (* P ≤ 0.05; ** P ≤ 0.01).
    Figure Legend Snippet: Localisation and expression of E-cadherin. ( A ) Immunofluorescence images of E-cadherin expression in green and DNA staining (DAPI) in blue. A secondary antibody control (AlexaFluor 488) for P cells is included. In CSZ, polygonal population is indicated with an asterisk and spindled cells with and an arrow. Scale bar: 40 µm. ( B ) A representative experiment of E-cadherin expression by Western Blot and densitometry graphics corresponding to three independent experiments are showed (Mean ± SD). Load control: β-actin. Separate gels where used for cell line and tumour cells. ROD (Relative optic density). ( C ) mRNA levels resulting from RT-PCR analysis. Relative data to their corresponding P or P T population are presented in the graph. (* P ≤ 0.05; ** P ≤ 0.01).

    Techniques Used: Expressing, Immunofluorescence, Staining, Western Blot, Reverse Transcription Polymerase Chain Reaction

    14) Product Images from "High-yield fabrication of DNA and RNA constructs for single molecule force and torque spectroscopy experiments"

    Article Title: High-yield fabrication of DNA and RNA constructs for single molecule force and torque spectroscopy experiments

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkz851

    Experimental strategies to assemble long DNA and RNA hairpins. The colored lines represent different nucleic acid strands. BIO and DIG are respectively biotin- and digoxygenin-labeled. ( A ) DNA hairpin construct using LNC: linear or plasmid DNA is used as template for PCR reactions; amplified fragments are purified and digested; fragments are then submitted to three rounds of purification and ligation (L1, L2, L3) to obtain the desired final product. ( B ) DNA hairpin construct, annealing method (ANC): template DNA is amplified by PCR and purified (pur.); one strand of the amplified fragments is nicked with enzymes Nb.BbvCI or Nt.BbvCI, gel purified and annealed (ann.) to obtain the final construct. ( C ) RNA hairpin construct: template DNA is amplified by PCR and purified, stem is amplified in three separate parts; RNA products are obtained by IVTR, purified and monophosphorylated (mP); products are then annealed and ligated to obtained the final construct.
    Figure Legend Snippet: Experimental strategies to assemble long DNA and RNA hairpins. The colored lines represent different nucleic acid strands. BIO and DIG are respectively biotin- and digoxygenin-labeled. ( A ) DNA hairpin construct using LNC: linear or plasmid DNA is used as template for PCR reactions; amplified fragments are purified and digested; fragments are then submitted to three rounds of purification and ligation (L1, L2, L3) to obtain the desired final product. ( B ) DNA hairpin construct, annealing method (ANC): template DNA is amplified by PCR and purified (pur.); one strand of the amplified fragments is nicked with enzymes Nb.BbvCI or Nt.BbvCI, gel purified and annealed (ann.) to obtain the final construct. ( C ) RNA hairpin construct: template DNA is amplified by PCR and purified, stem is amplified in three separate parts; RNA products are obtained by IVTR, purified and monophosphorylated (mP); products are then annealed and ligated to obtained the final construct.

    Techniques Used: Labeling, Construct, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Purification, Ligation

    Experimental strategies to assemble linear DNA and RNA constructs. The colored lines represent different nucleic acid strands. BIO and DIG are respectively biotin- and digoxygenin-labeled ( A ) DNA construct, ligation method (LNC): linear or plasmid DNA is used as a template for restriction digestions and PCR reactions; fragments are purified (pur.) and ligated (lig.) to obtain the desired final product. ( B ) DNA construct, annealing method (ANC): plasmid DNA is used as template for PCR reactions; one strand is nicked and removed; complementary single strands are annealed (ann.) to obtain the desired final product. ( C ) RNA construct, coilable (ANC): RNA strands are obtained by run-off in vitro transcription reaction (IVTR), then purified and annealed. Single strands are monophosphorylated (mP) prior to annealing and then ligated (lig.) to obtain a coilable product. ( D ) RNA construct, non-coilable (ANC): template DNA is amplified by PCR and purified; RNA single strands are obtained as in (C) and annealed.
    Figure Legend Snippet: Experimental strategies to assemble linear DNA and RNA constructs. The colored lines represent different nucleic acid strands. BIO and DIG are respectively biotin- and digoxygenin-labeled ( A ) DNA construct, ligation method (LNC): linear or plasmid DNA is used as a template for restriction digestions and PCR reactions; fragments are purified (pur.) and ligated (lig.) to obtain the desired final product. ( B ) DNA construct, annealing method (ANC): plasmid DNA is used as template for PCR reactions; one strand is nicked and removed; complementary single strands are annealed (ann.) to obtain the desired final product. ( C ) RNA construct, coilable (ANC): RNA strands are obtained by run-off in vitro transcription reaction (IVTR), then purified and annealed. Single strands are monophosphorylated (mP) prior to annealing and then ligated (lig.) to obtain a coilable product. ( D ) RNA construct, non-coilable (ANC): template DNA is amplified by PCR and purified; RNA single strands are obtained as in (C) and annealed.

    Techniques Used: Construct, Labeling, Ligation, Plasmid Preparation, Polymerase Chain Reaction, Purification, In Vitro, Amplification

    15) Product Images from "DNA polymerase stalling at structured DNA constrains the expansion of short tandem repeats"

    Article Title: DNA polymerase stalling at structured DNA constrains the expansion of short tandem repeats

    Journal: Genome Biology

    doi: 10.1186/s13059-020-02124-x

    Pooled measurement of DNA polymerase stalling at STRs. a Overview of the high-throughput primer extension assay used to monitor DNA synthesis at designed sequences. A library of 20,000 sequences comprising all STR permutations at three different lengths together with control structured DNA sequences was synthesised on a programmable microarray, eluted and inserted into a phagemid vector. After PCR amplification, insertion into a phagemid vector and bacterial amplification, circular single-stranded DNA templates were produced using a M13KO7 helper phage. Fluorescently labelled primer (P3) and structure annealing were performed before initiating DNA synthesis through the addition of T7 DNA polymerase. Primers are then either fully extended to the length of the circular template, or the extension is stopped within STRs if the DNA polymerases stall at structured DNAs. b Extended and stalled products were then analysed by denaturing poly acrylamide gel electrophoresis (PAGE), recovered from the gel matrix and prepared for high-throughput sequencing. DNA polymerase stalling was then quantified by analysing the enrichment of each sequence from the library in the stalled and extended fractions. Representative fluorescence gel imaging of primer extension reactions on templates containing a G-quadruplex (G4) structure, a mutated G4 or the entire DNA library, stopped after the indicated times, is reported for comparison. Blue and red arrows indicate the position of the extended and stalled products respectively. The green line highlights the presence of transient stall sites that disappear over time
    Figure Legend Snippet: Pooled measurement of DNA polymerase stalling at STRs. a Overview of the high-throughput primer extension assay used to monitor DNA synthesis at designed sequences. A library of 20,000 sequences comprising all STR permutations at three different lengths together with control structured DNA sequences was synthesised on a programmable microarray, eluted and inserted into a phagemid vector. After PCR amplification, insertion into a phagemid vector and bacterial amplification, circular single-stranded DNA templates were produced using a M13KO7 helper phage. Fluorescently labelled primer (P3) and structure annealing were performed before initiating DNA synthesis through the addition of T7 DNA polymerase. Primers are then either fully extended to the length of the circular template, or the extension is stopped within STRs if the DNA polymerases stall at structured DNAs. b Extended and stalled products were then analysed by denaturing poly acrylamide gel electrophoresis (PAGE), recovered from the gel matrix and prepared for high-throughput sequencing. DNA polymerase stalling was then quantified by analysing the enrichment of each sequence from the library in the stalled and extended fractions. Representative fluorescence gel imaging of primer extension reactions on templates containing a G-quadruplex (G4) structure, a mutated G4 or the entire DNA library, stopped after the indicated times, is reported for comparison. Blue and red arrows indicate the position of the extended and stalled products respectively. The green line highlights the presence of transient stall sites that disappear over time

    Techniques Used: High Throughput Screening Assay, Primer Extension Assay, DNA Synthesis, Microarray, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Produced, Polyacrylamide Gel Electrophoresis, Next-Generation Sequencing, Sequencing, Fluorescence, Imaging

    16) Product Images from "DNA polymerase stalling at structured DNA constrains the expansion of Short Tandem Repeats"

    Article Title: DNA polymerase stalling at structured DNA constrains the expansion of Short Tandem Repeats

    Journal: bioRxiv

    doi: 10.1101/2020.06.20.162743

    Pooled measurement of DNA polymerase stalling at STRs. (a) Overview of the high-throughput primer extension assay used to monitor DNA synthesis at designed sequences. A library of 20,000 sequences comprising all STR permutations at three different lengths together with control structured DNA sequences was synthesised on a programmable microarray, eluted and inserted into a phagemid vector. After PCR amplification, insertion into a phagemid vector and bacterial amplification, circular single-stranded DNA templates were produced using a M13KO7 helper phage. Fluorescently labelled primer (P3) and structures annealing were performed before initiating DNA synthesis through the addition of T7 DNA polymerase. Primers are then either fully extended to the length of the circular template or the extension is stopped within STRs if the DNA polymerases stall at structured DNAs. (b) Extended and stalled products were then analysed by denaturing Poly Acrylamide Gel (PAGE) electrophoresis, recovered from the gel matrix and prepared for high throughput sequencing. DNA polymerase stalling was then quantified by analysing the enrichment of each sequence form the library in the stalled and extended fractions. Representative fluorescence gel imaging of primer extension reactions on templates containing a G-quadruplex (G4) structure, a mutated G4 or the entire DNA library, stopped after the indicated times, are reported for comparison. Blue and red arrows indicate the position of the extended and stalled products respectively. The green line highlights the presence of transient stall sites that disappear overtime.
    Figure Legend Snippet: Pooled measurement of DNA polymerase stalling at STRs. (a) Overview of the high-throughput primer extension assay used to monitor DNA synthesis at designed sequences. A library of 20,000 sequences comprising all STR permutations at three different lengths together with control structured DNA sequences was synthesised on a programmable microarray, eluted and inserted into a phagemid vector. After PCR amplification, insertion into a phagemid vector and bacterial amplification, circular single-stranded DNA templates were produced using a M13KO7 helper phage. Fluorescently labelled primer (P3) and structures annealing were performed before initiating DNA synthesis through the addition of T7 DNA polymerase. Primers are then either fully extended to the length of the circular template or the extension is stopped within STRs if the DNA polymerases stall at structured DNAs. (b) Extended and stalled products were then analysed by denaturing Poly Acrylamide Gel (PAGE) electrophoresis, recovered from the gel matrix and prepared for high throughput sequencing. DNA polymerase stalling was then quantified by analysing the enrichment of each sequence form the library in the stalled and extended fractions. Representative fluorescence gel imaging of primer extension reactions on templates containing a G-quadruplex (G4) structure, a mutated G4 or the entire DNA library, stopped after the indicated times, are reported for comparison. Blue and red arrows indicate the position of the extended and stalled products respectively. The green line highlights the presence of transient stall sites that disappear overtime.

    Techniques Used: High Throughput Screening Assay, Primer Extension Assay, DNA Synthesis, Microarray, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Produced, Acrylamide Gel Assay, Polyacrylamide Gel Electrophoresis, Electrophoresis, Next-Generation Sequencing, Sequencing, Fluorescence, Imaging

    17) Product Images from "DNA polymerase stalling at structured DNA constrains the expansion of Short Tandem Repeats"

    Article Title: DNA polymerase stalling at structured DNA constrains the expansion of Short Tandem Repeats

    Journal: bioRxiv

    doi: 10.1101/2020.06.20.162743

    Pooled measurement of DNA polymerase stalling at STRs. (a) Overview of the high-throughput primer extension assay used to monitor DNA synthesis at designed sequences. A library of 20,000 sequences comprising all STR permutations at three different lengths together with control structured DNA sequences was synthesised on a programmable microarray, eluted and inserted into a phagemid vector. After PCR amplification, insertion into a phagemid vector and bacterial amplification, circular single-stranded DNA templates were produced using a M13KO7 helper phage. Fluorescently labelled primer (P3) and structures annealing were performed before initiating DNA synthesis through the addition of T7 DNA polymerase. Primers are then either fully extended to the length of the circular template or the extension is stopped within STRs if the DNA polymerases stall at structured DNAs. (b) Extended and stalled products were then analysed by denaturing Poly Acrylamide Gel (PAGE) electrophoresis, recovered from the gel matrix and prepared for high throughput sequencing. DNA polymerase stalling was then quantified by analysing the enrichment of each sequence form the library in the stalled and extended fractions. Representative fluorescence gel imaging of primer extension reactions on templates containing a G-quadruplex (G4) structure, a mutated G4 or the entire DNA library, stopped after the indicated times, are reported for comparison. Blue and red arrows indicate the position of the extended and stalled products respectively. The green line highlights the presence of transient stall sites that disappear overtime.
    Figure Legend Snippet: Pooled measurement of DNA polymerase stalling at STRs. (a) Overview of the high-throughput primer extension assay used to monitor DNA synthesis at designed sequences. A library of 20,000 sequences comprising all STR permutations at three different lengths together with control structured DNA sequences was synthesised on a programmable microarray, eluted and inserted into a phagemid vector. After PCR amplification, insertion into a phagemid vector and bacterial amplification, circular single-stranded DNA templates were produced using a M13KO7 helper phage. Fluorescently labelled primer (P3) and structures annealing were performed before initiating DNA synthesis through the addition of T7 DNA polymerase. Primers are then either fully extended to the length of the circular template or the extension is stopped within STRs if the DNA polymerases stall at structured DNAs. (b) Extended and stalled products were then analysed by denaturing Poly Acrylamide Gel (PAGE) electrophoresis, recovered from the gel matrix and prepared for high throughput sequencing. DNA polymerase stalling was then quantified by analysing the enrichment of each sequence form the library in the stalled and extended fractions. Representative fluorescence gel imaging of primer extension reactions on templates containing a G-quadruplex (G4) structure, a mutated G4 or the entire DNA library, stopped after the indicated times, are reported for comparison. Blue and red arrows indicate the position of the extended and stalled products respectively. The green line highlights the presence of transient stall sites that disappear overtime.

    Techniques Used: High Throughput Screening Assay, Primer Extension Assay, DNA Synthesis, Microarray, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Produced, Acrylamide Gel Assay, Polyacrylamide Gel Electrophoresis, Electrophoresis, Next-Generation Sequencing, Sequencing, Fluorescence, Imaging

    18) Product Images from "Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application"

    Article Title: Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application

    Journal: BMC Genomics

    doi: 10.1186/s12864-017-4371-5

    Storage condition of purified ChIP DNA is important. Purified ChIP DNA was adjusted to a concentration of 1 ng/μL ( a ) or 0.1 ng/μL ( b ), aliquoted into 4 different types of microcentrifuge tubes in 15 μL volume, and stored at −20 °C. DNA was quantified using Qubit dsDNA High Sensitivity assay at the indicated time points and expressed as a percentage of the amount measured at day 0. Three independent DNA samples were used in the experiment and DNA concentration from five tubes were measured at each time point. MaxyClear, Axygen® 1.7 mL MaxyClear Snaplock Microcentrifuge Tube; LoBind, Eppendorf DNA LoBind Snap Cap PCR Tube; Siliconized, Fisherbrand™ Siliconized Low-Retention Microcentrifuge Tube; Premium, Fisherbrand™ Premium Microcentrifuge Tube
    Figure Legend Snippet: Storage condition of purified ChIP DNA is important. Purified ChIP DNA was adjusted to a concentration of 1 ng/μL ( a ) or 0.1 ng/μL ( b ), aliquoted into 4 different types of microcentrifuge tubes in 15 μL volume, and stored at −20 °C. DNA was quantified using Qubit dsDNA High Sensitivity assay at the indicated time points and expressed as a percentage of the amount measured at day 0. Three independent DNA samples were used in the experiment and DNA concentration from five tubes were measured at each time point. MaxyClear, Axygen® 1.7 mL MaxyClear Snaplock Microcentrifuge Tube; LoBind, Eppendorf DNA LoBind Snap Cap PCR Tube; Siliconized, Fisherbrand™ Siliconized Low-Retention Microcentrifuge Tube; Premium, Fisherbrand™ Premium Microcentrifuge Tube

    Techniques Used: Purification, Chromatin Immunoprecipitation, Concentration Assay, Sensitive Assay, Polymerase Chain Reaction

    DNA purification reagents vary in their ability to recover low amounts of DNA from de-crosslinked chromatin. a Recovered DNA amount by different DNA purification reagents from de-crosslinked chromatin. De-crosslinked chromatin estimated to include 1 ng range DNA in ChIP elution buffer was purified following the manufacturer’s instructions. The data were generated from triplicate DNA samples derived from three independent preparations. Zy, ChIP DNA Clean Concentrator™ (Zymo Research); Pr, Wizard® SV Gel and PCR Clean-Up System (Promega); Th, GeneJET PCR Purification Kit (Thermo Fisher Scientific); In, PureLink® PCR Purification Kit (Invitrogen); Ne, Monarch® PCR DNA Cleanup Kit (New England Biolabs); Am, Chromatin IP DNA Purification Kit (Active Motif); Qp, QIAquick PCR Purification Kit (Qiagen); Qm, MinElute PCR Purification Kit (Qiagen); Ba, Agencourt AMPure XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); Br, RNAClean™ XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); PC, phenol/chloroform extraction. b Interference of PCR amplification by purified eluent of purification reagents. 9 μL eluent was mixed with 1 μL 166 bp of Drosophila probe DNA (0.0001 ng), and the resulting mixture was used as the template in 20 μl of real-time PCR reaction. The Ct value for Drosophila probe DNA from TE buffer was set as 100%. The experiment was repeated 3 times using de-crosslinked chromatin estimated to include 1 ng of DNA. c Size profiles of DNA purified by different reagents. The DNAs purified from de-crosslinked chromatin estimated to include 50 ng range DNA was analyzed by AATI Fragment Analyzer. DNA size (bp) is shown
    Figure Legend Snippet: DNA purification reagents vary in their ability to recover low amounts of DNA from de-crosslinked chromatin. a Recovered DNA amount by different DNA purification reagents from de-crosslinked chromatin. De-crosslinked chromatin estimated to include 1 ng range DNA in ChIP elution buffer was purified following the manufacturer’s instructions. The data were generated from triplicate DNA samples derived from three independent preparations. Zy, ChIP DNA Clean Concentrator™ (Zymo Research); Pr, Wizard® SV Gel and PCR Clean-Up System (Promega); Th, GeneJET PCR Purification Kit (Thermo Fisher Scientific); In, PureLink® PCR Purification Kit (Invitrogen); Ne, Monarch® PCR DNA Cleanup Kit (New England Biolabs); Am, Chromatin IP DNA Purification Kit (Active Motif); Qp, QIAquick PCR Purification Kit (Qiagen); Qm, MinElute PCR Purification Kit (Qiagen); Ba, Agencourt AMPure XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); Br, RNAClean™ XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); PC, phenol/chloroform extraction. b Interference of PCR amplification by purified eluent of purification reagents. 9 μL eluent was mixed with 1 μL 166 bp of Drosophila probe DNA (0.0001 ng), and the resulting mixture was used as the template in 20 μl of real-time PCR reaction. The Ct value for Drosophila probe DNA from TE buffer was set as 100%. The experiment was repeated 3 times using de-crosslinked chromatin estimated to include 1 ng of DNA. c Size profiles of DNA purified by different reagents. The DNAs purified from de-crosslinked chromatin estimated to include 50 ng range DNA was analyzed by AATI Fragment Analyzer. DNA size (bp) is shown

    Techniques Used: DNA Purification, Chromatin Immunoprecipitation, Purification, Generated, Derivative Assay, Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction

    19) Product Images from "Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application"

    Article Title: Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application

    Journal: BMC Genomics

    doi: 10.1186/s12864-017-4371-5

    Storage condition of purified ChIP DNA is important. Purified ChIP DNA was adjusted to a concentration of 1 ng/μL ( a ) or 0.1 ng/μL ( b ), aliquoted into 4 different types of microcentrifuge tubes in 15 μL volume, and stored at −20 °C. DNA was quantified using Qubit dsDNA High Sensitivity assay at the indicated time points and expressed as a percentage of the amount measured at day 0. Three independent DNA samples were used in the experiment and DNA concentration from five tubes were measured at each time point. MaxyClear, Axygen® 1.7 mL MaxyClear Snaplock Microcentrifuge Tube; LoBind, Eppendorf DNA LoBind Snap Cap PCR Tube; Siliconized, Fisherbrand™ Siliconized Low-Retention Microcentrifuge Tube; Premium, Fisherbrand™ Premium Microcentrifuge Tube
    Figure Legend Snippet: Storage condition of purified ChIP DNA is important. Purified ChIP DNA was adjusted to a concentration of 1 ng/μL ( a ) or 0.1 ng/μL ( b ), aliquoted into 4 different types of microcentrifuge tubes in 15 μL volume, and stored at −20 °C. DNA was quantified using Qubit dsDNA High Sensitivity assay at the indicated time points and expressed as a percentage of the amount measured at day 0. Three independent DNA samples were used in the experiment and DNA concentration from five tubes were measured at each time point. MaxyClear, Axygen® 1.7 mL MaxyClear Snaplock Microcentrifuge Tube; LoBind, Eppendorf DNA LoBind Snap Cap PCR Tube; Siliconized, Fisherbrand™ Siliconized Low-Retention Microcentrifuge Tube; Premium, Fisherbrand™ Premium Microcentrifuge Tube

    Techniques Used: Purification, Chromatin Immunoprecipitation, Concentration Assay, Sensitive Assay, Polymerase Chain Reaction

    DNA purification reagents vary in their ability to recover low amounts of DNA from de-crosslinked chromatin. a Recovered DNA amount by different DNA purification reagents from de-crosslinked chromatin. De-crosslinked chromatin estimated to include 1 ng range DNA in ChIP elution buffer was purified following the manufacturer’s instructions. The data were generated from triplicate DNA samples derived from three independent preparations. Zy, ChIP DNA Clean Concentrator™ (Zymo Research); Pr, Wizard® SV Gel and PCR Clean-Up System (Promega); Th, GeneJET PCR Purification Kit (Thermo Fisher Scientific); In, PureLink® PCR Purification Kit (Invitrogen); Ne, Monarch® PCR DNA Cleanup Kit (New England Biolabs); Am, Chromatin IP DNA Purification Kit (Active Motif); Qp, QIAquick PCR Purification Kit (Qiagen); Qm, MinElute PCR Purification Kit (Qiagen); Ba, Agencourt AMPure XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); Br, RNAClean™ XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); PC, phenol/chloroform extraction. b Interference of PCR amplification by purified eluent of purification reagents. 9 μL eluent was mixed with 1 μL 166 bp of Drosophila probe DNA (0.0001 ng), and the resulting mixture was used as the template in 20 μl of real-time PCR reaction. The Ct value for Drosophila probe DNA from TE buffer was set as 100%. The experiment was repeated 3 times using de-crosslinked chromatin estimated to include 1 ng of DNA. c Size profiles of DNA purified by different reagents. The DNAs purified from de-crosslinked chromatin estimated to include 50 ng range DNA was analyzed by AATI Fragment Analyzer. DNA size (bp) is shown
    Figure Legend Snippet: DNA purification reagents vary in their ability to recover low amounts of DNA from de-crosslinked chromatin. a Recovered DNA amount by different DNA purification reagents from de-crosslinked chromatin. De-crosslinked chromatin estimated to include 1 ng range DNA in ChIP elution buffer was purified following the manufacturer’s instructions. The data were generated from triplicate DNA samples derived from three independent preparations. Zy, ChIP DNA Clean Concentrator™ (Zymo Research); Pr, Wizard® SV Gel and PCR Clean-Up System (Promega); Th, GeneJET PCR Purification Kit (Thermo Fisher Scientific); In, PureLink® PCR Purification Kit (Invitrogen); Ne, Monarch® PCR DNA Cleanup Kit (New England Biolabs); Am, Chromatin IP DNA Purification Kit (Active Motif); Qp, QIAquick PCR Purification Kit (Qiagen); Qm, MinElute PCR Purification Kit (Qiagen); Ba, Agencourt AMPure XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); Br, RNAClean™ XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); PC, phenol/chloroform extraction. b Interference of PCR amplification by purified eluent of purification reagents. 9 μL eluent was mixed with 1 μL 166 bp of Drosophila probe DNA (0.0001 ng), and the resulting mixture was used as the template in 20 μl of real-time PCR reaction. The Ct value for Drosophila probe DNA from TE buffer was set as 100%. The experiment was repeated 3 times using de-crosslinked chromatin estimated to include 1 ng of DNA. c Size profiles of DNA purified by different reagents. The DNAs purified from de-crosslinked chromatin estimated to include 50 ng range DNA was analyzed by AATI Fragment Analyzer. DNA size (bp) is shown

    Techniques Used: DNA Purification, Chromatin Immunoprecipitation, Purification, Generated, Derivative Assay, Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction

    20) Product Images from "Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis"

    Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky067

    Darwin Assembly using a θ oligonucleotide. Here, a single θ oligonucleotide is used in place of the two boundary oligonucleotides allowing enzymatic cleanup after the assembly reaction. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the nicked strand degraded by exonuclease III (1). Inner oligonucleotides and a single θ oligonucleotide are annealed to the ssDNA plasmid (2). The θ oligonucleotide encodes both assembly priming and termination sequences linked by a flexible linker such that successful assembly of the mutated strand results in a closed circle (3). The template plasmid can now be linearized (e.g. at the yellow dot, by adding a targeting oligonucleotide and appropriate restriction endonuclease) and both exonuclease I and exonuclease III added to degrade any non-circular DNA (4). The mutated gene can now be amplified from the closed circle by PCR (5) and cloned into a fresh vector (6) using the type IIS restriction sites (white dots).
    Figure Legend Snippet: Darwin Assembly using a θ oligonucleotide. Here, a single θ oligonucleotide is used in place of the two boundary oligonucleotides allowing enzymatic cleanup after the assembly reaction. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the nicked strand degraded by exonuclease III (1). Inner oligonucleotides and a single θ oligonucleotide are annealed to the ssDNA plasmid (2). The θ oligonucleotide encodes both assembly priming and termination sequences linked by a flexible linker such that successful assembly of the mutated strand results in a closed circle (3). The template plasmid can now be linearized (e.g. at the yellow dot, by adding a targeting oligonucleotide and appropriate restriction endonuclease) and both exonuclease I and exonuclease III added to degrade any non-circular DNA (4). The mutated gene can now be amplified from the closed circle by PCR (5) and cloned into a fresh vector (6) using the type IIS restriction sites (white dots).

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

    Darwin assembled TgoT DNA polymerase library. ( A ) Five separate sequencing reactions (range and reads shown in blue) were required to sample the diversity introduced across the eight target residues (shown in red along the TgoT gene). Mutations included focused degeneracies (e.g. YWC used against Y384) or ‘small intelligent’ (S-int) diversity (NDT, VMA, ATG and TGG oligonucleotides mixed in a 12:6:1:1 ratio). Resulting incorporation is shown in box plots with outliers explicitly labelled. Wild-type contamination was determined from positions where diversity excluded those sequences (N.A.: not applicable). As with the T7 RNA polymerase library, incorporation trends and biases were analysed to identify any biases in assembly. Ranked incorporation frequencies are shown for the residues targeted with ‘small intelligent’ diversity, and the top three highest (greens) and lowest (oranges) ranked codons (based on a straight sum of ranks) are highlighted ( B ). Outnest PCR of the TgoT DNA polymerase library (expected product of 2501 bp) showing that the final PCR can be carried out with either A- (MyTaq) or B-family (Q5) polymerases ( C ). MW: 1 kb ladder (NEB). NT: no template PCR control.
    Figure Legend Snippet: Darwin assembled TgoT DNA polymerase library. ( A ) Five separate sequencing reactions (range and reads shown in blue) were required to sample the diversity introduced across the eight target residues (shown in red along the TgoT gene). Mutations included focused degeneracies (e.g. YWC used against Y384) or ‘small intelligent’ (S-int) diversity (NDT, VMA, ATG and TGG oligonucleotides mixed in a 12:6:1:1 ratio). Resulting incorporation is shown in box plots with outliers explicitly labelled. Wild-type contamination was determined from positions where diversity excluded those sequences (N.A.: not applicable). As with the T7 RNA polymerase library, incorporation trends and biases were analysed to identify any biases in assembly. Ranked incorporation frequencies are shown for the residues targeted with ‘small intelligent’ diversity, and the top three highest (greens) and lowest (oranges) ranked codons (based on a straight sum of ranks) are highlighted ( B ). Outnest PCR of the TgoT DNA polymerase library (expected product of 2501 bp) showing that the final PCR can be carried out with either A- (MyTaq) or B-family (Q5) polymerases ( C ). MW: 1 kb ladder (NEB). NT: no template PCR control.

    Techniques Used: Sequencing, Polymerase Chain Reaction

    Principles of Darwin Assembly. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the cut strand degraded by exonuclease III (1). Boundary and inner (mutagenic) oligonucleotides are annealed to the ssDNA plasmid (2). Key features of the oligonucleotides are highlighted: 5′-boundary oligonucleotide is 5′-biotinylated; non-complementary overhangs are shown in blue with Type IIs endonuclease recognition sites shown in white; mutations are shown as red X in the inner oligonucleotides; 3′-boundary oligonucleotide is protected at its 3′-end. After annealing, primers are extended and ligated in an isothermal assembly reaction (3). The assembled strand can be isolated by paramagnetic streptavidin-coated beads (4) and purified by alkali washing prior to PCR using outnested priming sites (5) and cloning (6) using the type IIS restriction sites (white dots). The purification step (4) is not always necessary but we found it improved PCR performance, especially for long assembly reactions ( > 1 kb).
    Figure Legend Snippet: Principles of Darwin Assembly. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the cut strand degraded by exonuclease III (1). Boundary and inner (mutagenic) oligonucleotides are annealed to the ssDNA plasmid (2). Key features of the oligonucleotides are highlighted: 5′-boundary oligonucleotide is 5′-biotinylated; non-complementary overhangs are shown in blue with Type IIs endonuclease recognition sites shown in white; mutations are shown as red X in the inner oligonucleotides; 3′-boundary oligonucleotide is protected at its 3′-end. After annealing, primers are extended and ligated in an isothermal assembly reaction (3). The assembled strand can be isolated by paramagnetic streptavidin-coated beads (4) and purified by alkali washing prior to PCR using outnested priming sites (5) and cloning (6) using the type IIS restriction sites (white dots). The purification step (4) is not always necessary but we found it improved PCR performance, especially for long assembly reactions ( > 1 kb).

    Techniques Used: Plasmid Preparation, Isolation, Purification, Polymerase Chain Reaction, Clone Assay

    21) Product Images from "Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis"

    Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky067

    Darwin Assembly using a θ oligonucleotide. Here, a single θ oligonucleotide is used in place of the two boundary oligonucleotides allowing enzymatic cleanup after the assembly reaction. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the nicked strand degraded by exonuclease III (1). Inner oligonucleotides and a single θ oligonucleotide are annealed to the ssDNA plasmid (2). The θ oligonucleotide encodes both assembly priming and termination sequences linked by a flexible linker such that successful assembly of the mutated strand results in a closed circle (3). The template plasmid can now be linearized (e.g. at the yellow dot, by adding a targeting oligonucleotide and appropriate restriction endonuclease) and both exonuclease I and exonuclease III added to degrade any non-circular DNA (4). The mutated gene can now be amplified from the closed circle by PCR (5) and cloned into a fresh vector (6) using the type IIS restriction sites (white dots).
    Figure Legend Snippet: Darwin Assembly using a θ oligonucleotide. Here, a single θ oligonucleotide is used in place of the two boundary oligonucleotides allowing enzymatic cleanup after the assembly reaction. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the nicked strand degraded by exonuclease III (1). Inner oligonucleotides and a single θ oligonucleotide are annealed to the ssDNA plasmid (2). The θ oligonucleotide encodes both assembly priming and termination sequences linked by a flexible linker such that successful assembly of the mutated strand results in a closed circle (3). The template plasmid can now be linearized (e.g. at the yellow dot, by adding a targeting oligonucleotide and appropriate restriction endonuclease) and both exonuclease I and exonuclease III added to degrade any non-circular DNA (4). The mutated gene can now be amplified from the closed circle by PCR (5) and cloned into a fresh vector (6) using the type IIS restriction sites (white dots).

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

    Darwin assembled TgoT DNA polymerase library. ( A ) Five separate sequencing reactions (range and reads shown in blue) were required to sample the diversity introduced across the eight target residues (shown in red along the TgoT gene). Mutations included focused degeneracies (e.g. YWC used against Y384) or ‘small intelligent’ (S-int) diversity (NDT, VMA, ATG and TGG oligonucleotides mixed in a 12:6:1:1 ratio). Resulting incorporation is shown in box plots with outliers explicitly labelled. Wild-type contamination was determined from positions where diversity excluded those sequences (N.A.: not applicable). As with the T7 RNA polymerase library, incorporation trends and biases were analysed to identify any biases in assembly. Ranked incorporation frequencies are shown for the residues targeted with ‘small intelligent’ diversity, and the top three highest (greens) and lowest (oranges) ranked codons (based on a straight sum of ranks) are highlighted ( B ). Outnest PCR of the TgoT DNA polymerase library (expected product of 2501 bp) showing that the final PCR can be carried out with either A- (MyTaq) or B-family (Q5) polymerases ( C ). MW: 1 kb ladder (NEB). NT: no template PCR control.
    Figure Legend Snippet: Darwin assembled TgoT DNA polymerase library. ( A ) Five separate sequencing reactions (range and reads shown in blue) were required to sample the diversity introduced across the eight target residues (shown in red along the TgoT gene). Mutations included focused degeneracies (e.g. YWC used against Y384) or ‘small intelligent’ (S-int) diversity (NDT, VMA, ATG and TGG oligonucleotides mixed in a 12:6:1:1 ratio). Resulting incorporation is shown in box plots with outliers explicitly labelled. Wild-type contamination was determined from positions where diversity excluded those sequences (N.A.: not applicable). As with the T7 RNA polymerase library, incorporation trends and biases were analysed to identify any biases in assembly. Ranked incorporation frequencies are shown for the residues targeted with ‘small intelligent’ diversity, and the top three highest (greens) and lowest (oranges) ranked codons (based on a straight sum of ranks) are highlighted ( B ). Outnest PCR of the TgoT DNA polymerase library (expected product of 2501 bp) showing that the final PCR can be carried out with either A- (MyTaq) or B-family (Q5) polymerases ( C ). MW: 1 kb ladder (NEB). NT: no template PCR control.

    Techniques Used: Sequencing, Polymerase Chain Reaction

    Principles of Darwin Assembly. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the cut strand degraded by exonuclease III (1). Boundary and inner (mutagenic) oligonucleotides are annealed to the ssDNA plasmid (2). Key features of the oligonucleotides are highlighted: 5′-boundary oligonucleotide is 5′-biotinylated; non-complementary overhangs are shown in blue with Type IIs endonuclease recognition sites shown in white; mutations are shown as red X in the inner oligonucleotides; 3′-boundary oligonucleotide is protected at its 3′-end. After annealing, primers are extended and ligated in an isothermal assembly reaction (3). The assembled strand can be isolated by paramagnetic streptavidin-coated beads (4) and purified by alkali washing prior to PCR using outnested priming sites (5) and cloning (6) using the type IIS restriction sites (white dots). The purification step (4) is not always necessary but we found it improved PCR performance, especially for long assembly reactions ( > 1 kb).
    Figure Legend Snippet: Principles of Darwin Assembly. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the cut strand degraded by exonuclease III (1). Boundary and inner (mutagenic) oligonucleotides are annealed to the ssDNA plasmid (2). Key features of the oligonucleotides are highlighted: 5′-boundary oligonucleotide is 5′-biotinylated; non-complementary overhangs are shown in blue with Type IIs endonuclease recognition sites shown in white; mutations are shown as red X in the inner oligonucleotides; 3′-boundary oligonucleotide is protected at its 3′-end. After annealing, primers are extended and ligated in an isothermal assembly reaction (3). The assembled strand can be isolated by paramagnetic streptavidin-coated beads (4) and purified by alkali washing prior to PCR using outnested priming sites (5) and cloning (6) using the type IIS restriction sites (white dots). The purification step (4) is not always necessary but we found it improved PCR performance, especially for long assembly reactions ( > 1 kb).

    Techniques Used: Plasmid Preparation, Isolation, Purification, Polymerase Chain Reaction, Clone Assay

    22) Product Images from "Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis"

    Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky067

    Darwin Assembly using a θ oligonucleotide. Here, a single θ oligonucleotide is used in place of the two boundary oligonucleotides allowing enzymatic cleanup after the assembly reaction. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the nicked strand degraded by exonuclease III (1). Inner oligonucleotides and a single θ oligonucleotide are annealed to the ssDNA plasmid (2). The θ oligonucleotide encodes both assembly priming and termination sequences linked by a flexible linker such that successful assembly of the mutated strand results in a closed circle (3). The template plasmid can now be linearized (e.g. at the yellow dot, by adding a targeting oligonucleotide and appropriate restriction endonuclease) and both exonuclease I and exonuclease III added to degrade any non-circular DNA (4). The mutated gene can now be amplified from the closed circle by PCR (5) and cloned into a fresh vector (6) using the type IIS restriction sites (white dots).
    Figure Legend Snippet: Darwin Assembly using a θ oligonucleotide. Here, a single θ oligonucleotide is used in place of the two boundary oligonucleotides allowing enzymatic cleanup after the assembly reaction. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the nicked strand degraded by exonuclease III (1). Inner oligonucleotides and a single θ oligonucleotide are annealed to the ssDNA plasmid (2). The θ oligonucleotide encodes both assembly priming and termination sequences linked by a flexible linker such that successful assembly of the mutated strand results in a closed circle (3). The template plasmid can now be linearized (e.g. at the yellow dot, by adding a targeting oligonucleotide and appropriate restriction endonuclease) and both exonuclease I and exonuclease III added to degrade any non-circular DNA (4). The mutated gene can now be amplified from the closed circle by PCR (5) and cloned into a fresh vector (6) using the type IIS restriction sites (white dots).

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

    Darwin assembled TgoT DNA polymerase library. ( A ) Five separate sequencing reactions (range and reads shown in blue) were required to sample the diversity introduced across the eight target residues (shown in red along the TgoT gene). Mutations included focused degeneracies (e.g. YWC used against Y384) or ‘small intelligent’ (S-int) diversity (NDT, VMA, ATG and TGG oligonucleotides mixed in a 12:6:1:1 ratio). Resulting incorporation is shown in box plots with outliers explicitly labelled. Wild-type contamination was determined from positions where diversity excluded those sequences (N.A.: not applicable). As with the T7 RNA polymerase library, incorporation trends and biases were analysed to identify any biases in assembly. Ranked incorporation frequencies are shown for the residues targeted with ‘small intelligent’ diversity, and the top three highest (greens) and lowest (oranges) ranked codons (based on a straight sum of ranks) are highlighted ( B ). Outnest PCR of the TgoT DNA polymerase library (expected product of 2501 bp) showing that the final PCR can be carried out with either A- (MyTaq) or B-family (Q5) polymerases ( C ). MW: 1 kb ladder (NEB). NT: no template PCR control.
    Figure Legend Snippet: Darwin assembled TgoT DNA polymerase library. ( A ) Five separate sequencing reactions (range and reads shown in blue) were required to sample the diversity introduced across the eight target residues (shown in red along the TgoT gene). Mutations included focused degeneracies (e.g. YWC used against Y384) or ‘small intelligent’ (S-int) diversity (NDT, VMA, ATG and TGG oligonucleotides mixed in a 12:6:1:1 ratio). Resulting incorporation is shown in box plots with outliers explicitly labelled. Wild-type contamination was determined from positions where diversity excluded those sequences (N.A.: not applicable). As with the T7 RNA polymerase library, incorporation trends and biases were analysed to identify any biases in assembly. Ranked incorporation frequencies are shown for the residues targeted with ‘small intelligent’ diversity, and the top three highest (greens) and lowest (oranges) ranked codons (based on a straight sum of ranks) are highlighted ( B ). Outnest PCR of the TgoT DNA polymerase library (expected product of 2501 bp) showing that the final PCR can be carried out with either A- (MyTaq) or B-family (Q5) polymerases ( C ). MW: 1 kb ladder (NEB). NT: no template PCR control.

    Techniques Used: Sequencing, Polymerase Chain Reaction

    Principles of Darwin Assembly. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the cut strand degraded by exonuclease III (1). Boundary and inner (mutagenic) oligonucleotides are annealed to the ssDNA plasmid (2). Key features of the oligonucleotides are highlighted: 5′-boundary oligonucleotide is 5′-biotinylated; non-complementary overhangs are shown in blue with Type IIs endonuclease recognition sites shown in white; mutations are shown as red X in the inner oligonucleotides; 3′-boundary oligonucleotide is protected at its 3′-end. After annealing, primers are extended and ligated in an isothermal assembly reaction (3). The assembled strand can be isolated by paramagnetic streptavidin-coated beads (4) and purified by alkali washing prior to PCR using outnested priming sites (5) and cloning (6) using the type IIS restriction sites (white dots). The purification step (4) is not always necessary but we found it improved PCR performance, especially for long assembly reactions ( > 1 kb).
    Figure Legend Snippet: Principles of Darwin Assembly. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the cut strand degraded by exonuclease III (1). Boundary and inner (mutagenic) oligonucleotides are annealed to the ssDNA plasmid (2). Key features of the oligonucleotides are highlighted: 5′-boundary oligonucleotide is 5′-biotinylated; non-complementary overhangs are shown in blue with Type IIs endonuclease recognition sites shown in white; mutations are shown as red X in the inner oligonucleotides; 3′-boundary oligonucleotide is protected at its 3′-end. After annealing, primers are extended and ligated in an isothermal assembly reaction (3). The assembled strand can be isolated by paramagnetic streptavidin-coated beads (4) and purified by alkali washing prior to PCR using outnested priming sites (5) and cloning (6) using the type IIS restriction sites (white dots). The purification step (4) is not always necessary but we found it improved PCR performance, especially for long assembly reactions ( > 1 kb).

    Techniques Used: Plasmid Preparation, Isolation, Purification, Polymerase Chain Reaction, Clone Assay

    23) Product Images from "EM-seq: Detection of DNA Methylation at Single Base Resolution from Picograms of DNA"

    Article Title: EM-seq: Detection of DNA Methylation at Single Base Resolution from Picograms of DNA

    Journal: bioRxiv

    doi: 10.1101/2019.12.20.884692

    Enzymatic Methyl-seq mechanism of action and workflow (A) Principle pathways that are important for enzymatic identification of 5mC and 5hmC using Enzymatic Methyl-seq. The actions of TET2 (blue) and T4-βGT (purple) on 5mC and its oxidation products, as well as the activity of APOBEC3A (green) on cytosine, 5gmC and 5caC are shown. The red cross represents no APOBEC3A activity. T4-βGT glucosylates 5hmC (pre-existing 5hmC and that formed by the action of TET2). TET2 converts 5mC through the intermediates 5hmC and 5fC into 5caC. APOBEC3A has limited activity on 5fC and undetectable activity on 5gmC and 5caC ( Figure 3C ). Uracil is replaced by thymine during PCR and is read as thymine during Illumina sequencing. (B) DNA is sheared to approximately 300 bp, end repaired and 3’ A-tailed. EM-seq adaptors are then ligated to the DNA. The DNA is treated with TET2 and T4-βGT before moving to the deamination reaction. The library is PCR amplified using EM-seq adaptor primers and can be sequenced on any Illumina sequencer.
    Figure Legend Snippet: Enzymatic Methyl-seq mechanism of action and workflow (A) Principle pathways that are important for enzymatic identification of 5mC and 5hmC using Enzymatic Methyl-seq. The actions of TET2 (blue) and T4-βGT (purple) on 5mC and its oxidation products, as well as the activity of APOBEC3A (green) on cytosine, 5gmC and 5caC are shown. The red cross represents no APOBEC3A activity. T4-βGT glucosylates 5hmC (pre-existing 5hmC and that formed by the action of TET2). TET2 converts 5mC through the intermediates 5hmC and 5fC into 5caC. APOBEC3A has limited activity on 5fC and undetectable activity on 5gmC and 5caC ( Figure 3C ). Uracil is replaced by thymine during PCR and is read as thymine during Illumina sequencing. (B) DNA is sheared to approximately 300 bp, end repaired and 3’ A-tailed. EM-seq adaptors are then ligated to the DNA. The DNA is treated with TET2 and T4-βGT before moving to the deamination reaction. The library is PCR amplified using EM-seq adaptor primers and can be sequenced on any Illumina sequencer.

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

    EM-seq Illumina libraries are superior to bisulfite libraries 10 ng, 50 ng or 200 ng of NA12878 DNA were spiked with control DNA (2 ng unmethylated lambda DNA and 0.1 ng CpG methylated pUC19) and Illumina libraries were made using either EM-seq or the Zymo Gold Bisulfite Kit prepped for Illumina sequencing using NEBNext Ultra II DNA library kit reagents. Libraries were sequenced on an Illumina NovaSeq 6000 and 324 M read pairs per library were used for analysis. (A) EM-seq uses less PCR cycles but results in more PCR product than whole genome bisulfite libraries (WGBS) for all NA12878 input amounts. (B) Table of sequencing and alignment metrics for EM-seq and WGBS libraries using 324 million Illumina read pairs. Metrics were calculated using bwa-meth, Samtools, and Picard. Theoretical coverage is calculated using the number of bases sequenced/total bases in the GRCh38 reference. Percent mapped refers to reads aligned to the reference genome (grch38+controls), Percent Dups refers to reads marked as duplicate by Picard MarkDuplicates, Percent Usable refers to the set of Proper-pair, MapQ 10+, Primary, non-Duplicate reads used in methylation calling (samtools view -F 0xF00 -q 10). Effective Coverage is the Percent Usable * Theoretical coverage. (C) GC-bias plot for EM-seq and WGBS libraries. EM-seq libraries display an even GC distribution while WGBS libraries have an AT-rich and GC-poor profile.
    Figure Legend Snippet: EM-seq Illumina libraries are superior to bisulfite libraries 10 ng, 50 ng or 200 ng of NA12878 DNA were spiked with control DNA (2 ng unmethylated lambda DNA and 0.1 ng CpG methylated pUC19) and Illumina libraries were made using either EM-seq or the Zymo Gold Bisulfite Kit prepped for Illumina sequencing using NEBNext Ultra II DNA library kit reagents. Libraries were sequenced on an Illumina NovaSeq 6000 and 324 M read pairs per library were used for analysis. (A) EM-seq uses less PCR cycles but results in more PCR product than whole genome bisulfite libraries (WGBS) for all NA12878 input amounts. (B) Table of sequencing and alignment metrics for EM-seq and WGBS libraries using 324 million Illumina read pairs. Metrics were calculated using bwa-meth, Samtools, and Picard. Theoretical coverage is calculated using the number of bases sequenced/total bases in the GRCh38 reference. Percent mapped refers to reads aligned to the reference genome (grch38+controls), Percent Dups refers to reads marked as duplicate by Picard MarkDuplicates, Percent Usable refers to the set of Proper-pair, MapQ 10+, Primary, non-Duplicate reads used in methylation calling (samtools view -F 0xF00 -q 10). Effective Coverage is the Percent Usable * Theoretical coverage. (C) GC-bias plot for EM-seq and WGBS libraries. EM-seq libraries display an even GC distribution while WGBS libraries have an AT-rich and GC-poor profile.

    Techniques Used: Lambda DNA Preparation, Methylation, Sequencing, Polymerase Chain Reaction

    24) Product Images from "Single telomere length analysis in Ustilago maydis, a high-resolution tool for examining fungal telomere length distribution and C-strand 5’-end processing"

    Article Title: Single telomere length analysis in Ustilago maydis, a high-resolution tool for examining fungal telomere length distribution and C-strand 5’-end processing

    Journal: Microbial Cell

    doi: 10.15698/mic2018.09.645

    FIGURE 1: STELA protocol and investigation of UT4/5-containing telomeres. (A) Schematic illustration of the structure of UT4 and UT5-containing telomeres in U. maydis . The use of telorette oligos to modify the C-strand and the use of primers (UT4/5-F and teltail) to generate STELA products are also illustrated. (B) Four individual STELA PCR reactions for UT4/5 telomeres were performed using 2.5 pg of ligated wild type DNA as the template and shown on the left. A parallel Southern analysis is shown on the right. The same UT4/5 subtelomeric probe was used to detect telomere fragments in both analyses. (C) STELA assays were performed using 5 pg wild type DNA as the template, and the UT4/5-F and teltail oligos as primers. Following gel electrophoresis and transfer to a nylon membrane, the products were first detected using a UT4/5 subtelomeric probe (left panel). Subsequently, the UT4/5 probe was stripped from the membrane and the products re-analyzed using a TTAGGG repeat probe (middle panel). The sizes of the STELA fragments in the middle panel were determined using TESLA software. The lengths of the telomere tracts were then calculated by subtracting the subtelomere length (~630 bp), and then plotted (right). Error bars designate standard error of means.
    Figure Legend Snippet: FIGURE 1: STELA protocol and investigation of UT4/5-containing telomeres. (A) Schematic illustration of the structure of UT4 and UT5-containing telomeres in U. maydis . The use of telorette oligos to modify the C-strand and the use of primers (UT4/5-F and teltail) to generate STELA products are also illustrated. (B) Four individual STELA PCR reactions for UT4/5 telomeres were performed using 2.5 pg of ligated wild type DNA as the template and shown on the left. A parallel Southern analysis is shown on the right. The same UT4/5 subtelomeric probe was used to detect telomere fragments in both analyses. (C) STELA assays were performed using 5 pg wild type DNA as the template, and the UT4/5-F and teltail oligos as primers. Following gel electrophoresis and transfer to a nylon membrane, the products were first detected using a UT4/5 subtelomeric probe (left panel). Subsequently, the UT4/5 probe was stripped from the membrane and the products re-analyzed using a TTAGGG repeat probe (middle panel). The sizes of the STELA fragments in the middle panel were determined using TESLA software. The lengths of the telomere tracts were then calculated by subtracting the subtelomere length (~630 bp), and then plotted (right). Error bars designate standard error of means.

    Techniques Used: Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Software

    25) Product Images from "Variability within rare cell states enables multiple paths towards drug resistance"

    Article Title: Variability within rare cell states enables multiple paths towards drug resistance

    Journal: bioRxiv

    doi: 10.1101/2020.03.18.996660

    Rewind identifies a distinct subpopulation of cells that require DOT1L inhibition to become vemurafenib resistant. A . Experimental approach for identifying the subpopulation of cells that require DOT1L inhibition to become vemurafenib resistant. These experiments began with approximately 400,000 WM989 A6-G3 cells transduced at an MOI ∼ 1.0 and allowed to divide for 6 days before splitting the culture into two groups. We treated one group with 4 μM DOT1L inhibitor (pinometostat) and the other with vehicle control (DMSO) for another 6 days. We then split each group again, fixing half as our “Carbon Copies” and treating the other half with 1 μM vemurafenib for ∼2.5 weeks. After vemurafenib treatment, we extracted genomic DNA from the remaining cells for barcode sequencing. B . We compared the abundance of each barcode identified in resistant cells pre-treated with DOT1L inhibitor versus resistant cells pre-treated with vehicle control as shown in A. This comparison revealed a subset of barcodes with a greater relative abundance in resistant cells pre-treated with DOT1L inhibitor than resistant cells pre-treated with vehicle control (blue points). We used these barcodes to design RNA FISH probes targeting cells requiring DOT1L inhibition to become vemurafenib resistant. A separate set of barcodes showed similar high abundance with or without DOT1L inhibition (orange points), which we used to design RNA FISH probes targeting primed cells not requiring DOT1L inhibition to become resistant. C . Using these probes, we labeled and sorted cells requiring DOT1L inhibition to become vemurafenib resistant (blue), primed cells not requiring DOT1L inhibition (orange), and non-primed cells (gray) from Carbon Copies for RNA sequencing. We separately sorted cells from Carbon Copies treated with DOT1L inhibitor and Carbon Copies treated with vehicle control (2 biological replicates each). D . To identify markers of cells that require DOT1L inhibition to become resistant, we used DESeq2 to compare their gene expression to non-primed cells (x-axis) and primed cells not requiring DOT1L inhibition (y-axis). In this analysis, we included cells sorted from all Carbon Copies (treated with DOT1L inhibitor or vehicle control) from 2 biological replicates and included DOT1L inhibitor treatment as a covariate in estimating log 2 fold changes. Red points correspond to genes differentially expressed in one or both comparisons (p-adjusted ≤0.1 and log 2 fold change ≥ 1). E . Expression of DEPTOR in transcripts per million (tpm) in the subpopulations isolated in B. Points indicate tpm values for experimental replicates. F . We used the same probe sets as in B. to identify cells in situ in Carbon Copies fixed prior to vemurafenib treatment, then measured single cell expression of DEPTOR, MGP, SOX10, MITF , and 6 priming markers by RNA FISH. Shown is the expression of DEPTOR in the indicated cell populations identified in the Carbon Copies treated with vehicle control. Each point corresponds to an individual cell. Error bars indicate 25th and 75th percentiles of distributions. Above each boxplot is the proportion of cells with levels of DEPTOR RNA above the indicated threshold (∼95th percentile in non-primed cells). G . We applied the UMAP algorithm to visualize the single cell expression data from in situ Carbon Copies. These plots include 423 cells from the vehicle control treated Carbon Copy. In the upper left plot, points are colored according to the fate of each cell as determined by its barcode. For the remaining plots points are colored by the expression level of the indicated gene in that cell. These data correspond to 1 of 2 biological replicates (See Supp. Fig 13 for additional replicate).
    Figure Legend Snippet: Rewind identifies a distinct subpopulation of cells that require DOT1L inhibition to become vemurafenib resistant. A . Experimental approach for identifying the subpopulation of cells that require DOT1L inhibition to become vemurafenib resistant. These experiments began with approximately 400,000 WM989 A6-G3 cells transduced at an MOI ∼ 1.0 and allowed to divide for 6 days before splitting the culture into two groups. We treated one group with 4 μM DOT1L inhibitor (pinometostat) and the other with vehicle control (DMSO) for another 6 days. We then split each group again, fixing half as our “Carbon Copies” and treating the other half with 1 μM vemurafenib for ∼2.5 weeks. After vemurafenib treatment, we extracted genomic DNA from the remaining cells for barcode sequencing. B . We compared the abundance of each barcode identified in resistant cells pre-treated with DOT1L inhibitor versus resistant cells pre-treated with vehicle control as shown in A. This comparison revealed a subset of barcodes with a greater relative abundance in resistant cells pre-treated with DOT1L inhibitor than resistant cells pre-treated with vehicle control (blue points). We used these barcodes to design RNA FISH probes targeting cells requiring DOT1L inhibition to become vemurafenib resistant. A separate set of barcodes showed similar high abundance with or without DOT1L inhibition (orange points), which we used to design RNA FISH probes targeting primed cells not requiring DOT1L inhibition to become resistant. C . Using these probes, we labeled and sorted cells requiring DOT1L inhibition to become vemurafenib resistant (blue), primed cells not requiring DOT1L inhibition (orange), and non-primed cells (gray) from Carbon Copies for RNA sequencing. We separately sorted cells from Carbon Copies treated with DOT1L inhibitor and Carbon Copies treated with vehicle control (2 biological replicates each). D . To identify markers of cells that require DOT1L inhibition to become resistant, we used DESeq2 to compare their gene expression to non-primed cells (x-axis) and primed cells not requiring DOT1L inhibition (y-axis). In this analysis, we included cells sorted from all Carbon Copies (treated with DOT1L inhibitor or vehicle control) from 2 biological replicates and included DOT1L inhibitor treatment as a covariate in estimating log 2 fold changes. Red points correspond to genes differentially expressed in one or both comparisons (p-adjusted ≤0.1 and log 2 fold change ≥ 1). E . Expression of DEPTOR in transcripts per million (tpm) in the subpopulations isolated in B. Points indicate tpm values for experimental replicates. F . We used the same probe sets as in B. to identify cells in situ in Carbon Copies fixed prior to vemurafenib treatment, then measured single cell expression of DEPTOR, MGP, SOX10, MITF , and 6 priming markers by RNA FISH. Shown is the expression of DEPTOR in the indicated cell populations identified in the Carbon Copies treated with vehicle control. Each point corresponds to an individual cell. Error bars indicate 25th and 75th percentiles of distributions. Above each boxplot is the proportion of cells with levels of DEPTOR RNA above the indicated threshold (∼95th percentile in non-primed cells). G . We applied the UMAP algorithm to visualize the single cell expression data from in situ Carbon Copies. These plots include 423 cells from the vehicle control treated Carbon Copy. In the upper left plot, points are colored according to the fate of each cell as determined by its barcode. For the remaining plots points are colored by the expression level of the indicated gene in that cell. These data correspond to 1 of 2 biological replicates (See Supp. Fig 13 for additional replicate).

    Techniques Used: Inhibition, Sequencing, Fluorescence In Situ Hybridization, Labeling, RNA Sequencing Assay, Expressing, Isolation, In Situ

    Rewind identifies rare cell states giving rise to vemurafenib resistant colonies. A . Schematic of Rewind approach for isolating the initial primed WM989 A6-G3 melanoma cells that ultimately give rise to vemurafenib resistant colonies. For the experiment shown, we transduced ∼ 200,000 WM989 A6-G3 cells at an MOI ∼ 1.0 with our Rewind barcode library. After 11 days (∼4 population doublings) we divided the culture in two, fixing half in suspension as a Carbon Copy and treating the other half with 1 μM vemurafenib to select for resistant cells. After 3 weeks in vemurafenib, we extracted genomic DNA from the resistant cells that remain and identified their Rewind barcodes by targeted sequencing. We then designed RNA FISH probes targeting 60 of these barcodes and used these probes to specifically label cells primed to become resistant from our Carbon Copy. We then sorted these cells out from the population, extracted cellular RNA and performed RNA sequencing. B . To assess the sensitivity and specificity of the Rewind experiment in A, we performed targeted sequencing to identify barcodes from cDNA generated during RNA-seq library preparation. Bar graphs show the abundance (y-axis) and rank (x-axis) of each sequenced barcode (≥ 5 normalized reads). Red bars correspond to barcodes targeted by our probe set and gray bars correspond to “off-target” barcode sequences. Inset shows the proportion of barcodes targeted by our probeset detected in each group. These data correspond to 1 of 2 replicates. In the second replicate, 30 out of 50 probed barcodes were detected in the sorted primed population. C . We performed differential expression analysis using DESeq2 of primed vs. non-primed sorted cells. Shown is the mean expression level (TPM) for protein coding genes in primed cells (y-axis) and log 2 fold change in expression estimated using DESeq2 (x-axis) compared to non-primed cells. Colors indicate differentially expressed genes related to ECM Organization and Cell Migration (red), MAPK and PI3K/Akt signalling pathways (blue) and previously identified resistance markers (purple; Shaffer et al. 2017). Genes were assigned to categories based on a consensus of KEGG pathway and GO enrichment analyses (See Methods for details). D . We selected the most differentially expressed, cell surface ECM-related gene ( ITGA3 ) to validate as a predictive marker of vemurafenib resistance in WM989 A6-G3. After staining cells with a fluorescently labelled antibody targeting ITGA3, we sorted the brightest 0.5% (ITGA3-High) and remaining (ITGA3-Low) populations, then treated both with 1 μM vemurafenib. After approximately 18 days, we fixed the cells, stained nuclei with DAPI then imaged the entire wells to quantify the number of resistant colonies and cells. The data correspond to 1 of 3 biological replicates (See Supp. Fig. 4 for additional replicates).
    Figure Legend Snippet: Rewind identifies rare cell states giving rise to vemurafenib resistant colonies. A . Schematic of Rewind approach for isolating the initial primed WM989 A6-G3 melanoma cells that ultimately give rise to vemurafenib resistant colonies. For the experiment shown, we transduced ∼ 200,000 WM989 A6-G3 cells at an MOI ∼ 1.0 with our Rewind barcode library. After 11 days (∼4 population doublings) we divided the culture in two, fixing half in suspension as a Carbon Copy and treating the other half with 1 μM vemurafenib to select for resistant cells. After 3 weeks in vemurafenib, we extracted genomic DNA from the resistant cells that remain and identified their Rewind barcodes by targeted sequencing. We then designed RNA FISH probes targeting 60 of these barcodes and used these probes to specifically label cells primed to become resistant from our Carbon Copy. We then sorted these cells out from the population, extracted cellular RNA and performed RNA sequencing. B . To assess the sensitivity and specificity of the Rewind experiment in A, we performed targeted sequencing to identify barcodes from cDNA generated during RNA-seq library preparation. Bar graphs show the abundance (y-axis) and rank (x-axis) of each sequenced barcode (≥ 5 normalized reads). Red bars correspond to barcodes targeted by our probe set and gray bars correspond to “off-target” barcode sequences. Inset shows the proportion of barcodes targeted by our probeset detected in each group. These data correspond to 1 of 2 replicates. In the second replicate, 30 out of 50 probed barcodes were detected in the sorted primed population. C . We performed differential expression analysis using DESeq2 of primed vs. non-primed sorted cells. Shown is the mean expression level (TPM) for protein coding genes in primed cells (y-axis) and log 2 fold change in expression estimated using DESeq2 (x-axis) compared to non-primed cells. Colors indicate differentially expressed genes related to ECM Organization and Cell Migration (red), MAPK and PI3K/Akt signalling pathways (blue) and previously identified resistance markers (purple; Shaffer et al. 2017). Genes were assigned to categories based on a consensus of KEGG pathway and GO enrichment analyses (See Methods for details). D . We selected the most differentially expressed, cell surface ECM-related gene ( ITGA3 ) to validate as a predictive marker of vemurafenib resistance in WM989 A6-G3. After staining cells with a fluorescently labelled antibody targeting ITGA3, we sorted the brightest 0.5% (ITGA3-High) and remaining (ITGA3-Low) populations, then treated both with 1 μM vemurafenib. After approximately 18 days, we fixed the cells, stained nuclei with DAPI then imaged the entire wells to quantify the number of resistant colonies and cells. The data correspond to 1 of 3 biological replicates (See Supp. Fig. 4 for additional replicates).

    Techniques Used: Sequencing, Fluorescence In Situ Hybridization, RNA Sequencing Assay, Generated, Expressing, Migration, Marker, Staining

    26) Product Images from "EM-seq: Detection of DNA Methylation at Single Base Resolution from Picograms of DNA"

    Article Title: EM-seq: Detection of DNA Methylation at Single Base Resolution from Picograms of DNA

    Journal: bioRxiv

    doi: 10.1101/2019.12.20.884692

    Enzymatic Methyl-seq mechanism of action and workflow (A) Principle pathways that are important for enzymatic identification of 5mC and 5hmC using Enzymatic Methyl-seq. The actions of TET2 (blue) and T4-βGT (purple) on 5mC and its oxidation products, as well as the activity of APOBEC3A (green) on cytosine, 5gmC and 5caC are shown. The red cross represents no APOBEC3A activity. T4-βGT glucosylates 5hmC (pre-existing 5hmC and that formed by the action of TET2). TET2 converts 5mC through the intermediates 5hmC and 5fC into 5caC. APOBEC3A has limited activity on 5fC and undetectable activity on 5gmC and 5caC ( Figure 3C ). Uracil is replaced by thymine during PCR and is read as thymine during Illumina sequencing. (B) DNA is sheared to approximately 300 bp, end repaired and 3’ A-tailed. EM-seq adaptors are then ligated to the DNA. The DNA is treated with TET2 and T4-βGT before moving to the deamination reaction. The library is PCR amplified using EM-seq adaptor primers and can be sequenced on any Illumina sequencer.
    Figure Legend Snippet: Enzymatic Methyl-seq mechanism of action and workflow (A) Principle pathways that are important for enzymatic identification of 5mC and 5hmC using Enzymatic Methyl-seq. The actions of TET2 (blue) and T4-βGT (purple) on 5mC and its oxidation products, as well as the activity of APOBEC3A (green) on cytosine, 5gmC and 5caC are shown. The red cross represents no APOBEC3A activity. T4-βGT glucosylates 5hmC (pre-existing 5hmC and that formed by the action of TET2). TET2 converts 5mC through the intermediates 5hmC and 5fC into 5caC. APOBEC3A has limited activity on 5fC and undetectable activity on 5gmC and 5caC ( Figure 3C ). Uracil is replaced by thymine during PCR and is read as thymine during Illumina sequencing. (B) DNA is sheared to approximately 300 bp, end repaired and 3’ A-tailed. EM-seq adaptors are then ligated to the DNA. The DNA is treated with TET2 and T4-βGT before moving to the deamination reaction. The library is PCR amplified using EM-seq adaptor primers and can be sequenced on any Illumina sequencer.

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

    EM-seq Illumina libraries are superior to bisulfite libraries 10 ng, 50 ng or 200 ng of NA12878 DNA were spiked with control DNA (2 ng unmethylated lambda DNA and 0.1 ng CpG methylated pUC19) and Illumina libraries were made using either EM-seq or the Zymo Gold Bisulfite Kit prepped for Illumina sequencing using NEBNext Ultra II DNA library kit reagents. Libraries were sequenced on an Illumina NovaSeq 6000 and 324 M read pairs per library were used for analysis. (A) EM-seq uses less PCR cycles but results in more PCR product than whole genome bisulfite libraries (WGBS) for all NA12878 input amounts. (B) Table of sequencing and alignment metrics for EM-seq and WGBS libraries using 324 million Illumina read pairs. Metrics were calculated using bwa-meth, Samtools, and Picard. Theoretical coverage is calculated using the number of bases sequenced/total bases in the GRCh38 reference. Percent mapped refers to reads aligned to the reference genome (grch38+controls), Percent Dups refers to reads marked as duplicate by Picard MarkDuplicates, Percent Usable refers to the set of Proper-pair, MapQ 10+, Primary, non-Duplicate reads used in methylation calling (samtools view -F 0xF00 -q 10). Effective Coverage is the Percent Usable * Theoretical coverage. (C) GC-bias plot for EM-seq and WGBS libraries. EM-seq libraries display an even GC distribution while WGBS libraries have an AT-rich and GC-poor profile.
    Figure Legend Snippet: EM-seq Illumina libraries are superior to bisulfite libraries 10 ng, 50 ng or 200 ng of NA12878 DNA were spiked with control DNA (2 ng unmethylated lambda DNA and 0.1 ng CpG methylated pUC19) and Illumina libraries were made using either EM-seq or the Zymo Gold Bisulfite Kit prepped for Illumina sequencing using NEBNext Ultra II DNA library kit reagents. Libraries were sequenced on an Illumina NovaSeq 6000 and 324 M read pairs per library were used for analysis. (A) EM-seq uses less PCR cycles but results in more PCR product than whole genome bisulfite libraries (WGBS) for all NA12878 input amounts. (B) Table of sequencing and alignment metrics for EM-seq and WGBS libraries using 324 million Illumina read pairs. Metrics were calculated using bwa-meth, Samtools, and Picard. Theoretical coverage is calculated using the number of bases sequenced/total bases in the GRCh38 reference. Percent mapped refers to reads aligned to the reference genome (grch38+controls), Percent Dups refers to reads marked as duplicate by Picard MarkDuplicates, Percent Usable refers to the set of Proper-pair, MapQ 10+, Primary, non-Duplicate reads used in methylation calling (samtools view -F 0xF00 -q 10). Effective Coverage is the Percent Usable * Theoretical coverage. (C) GC-bias plot for EM-seq and WGBS libraries. EM-seq libraries display an even GC distribution while WGBS libraries have an AT-rich and GC-poor profile.

    Techniques Used: Lambda DNA Preparation, Methylation, Sequencing, Polymerase Chain Reaction

    27) Product Images from "Fitness landscape of a dynamic RNA structure"

    Article Title: Fitness landscape of a dynamic RNA structure

    Journal: bioRxiv

    doi: 10.1101/2020.06.06.130575

    Generation of mutant library using site-saturation mutagenesis via a two-step PCR. Solid arrows denote oligonucleotides. In the first step, two pairs of oligonucleotides containing mixed bases ( https://www.idtdna.com/pages/products/custom-dna-rna/mixed-bases ) are used to amplify two separate fragments (purple and pink) containing one P1ex sub-region each. The 3’ end of the purple fragment and the 5’ end of the pink fragment share a 12-bp overlapping region, which allow self-annealing and subsequent 3’ extension during the second PCR. As a result, the assembled amplicons contain two varying sub-regions. The purple fragment is amplified using primers Frag1-f and Frag1-r, whereas the pink fragment is amplified using primers Frag2-f and Frag2-r ( Table S3 ).
    Figure Legend Snippet: Generation of mutant library using site-saturation mutagenesis via a two-step PCR. Solid arrows denote oligonucleotides. In the first step, two pairs of oligonucleotides containing mixed bases ( https://www.idtdna.com/pages/products/custom-dna-rna/mixed-bases ) are used to amplify two separate fragments (purple and pink) containing one P1ex sub-region each. The 3’ end of the purple fragment and the 5’ end of the pink fragment share a 12-bp overlapping region, which allow self-annealing and subsequent 3’ extension during the second PCR. As a result, the assembled amplicons contain two varying sub-regions. The purple fragment is amplified using primers Frag1-f and Frag1-r, whereas the pink fragment is amplified using primers Frag2-f and Frag2-r ( Table S3 ).

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Amplification

    28) Product Images from "DNA polymerase stalling at structured DNA constrains the expansion of Short Tandem Repeats"

    Article Title: DNA polymerase stalling at structured DNA constrains the expansion of Short Tandem Repeats

    Journal: bioRxiv

    doi: 10.1101/2020.06.20.162743

    Pooled measurement of DNA polymerase stalling at STRs. (a) Overview of the high-throughput primer extension assay used to monitor DNA synthesis at designed sequences. A library of 20,000 sequences comprising all STR permutations at three different lengths together with control structured DNA sequences was synthesised on a programmable microarray, eluted and inserted into a phagemid vector. After PCR amplification, insertion into a phagemid vector and bacterial amplification, circular single-stranded DNA templates were produced using a M13KO7 helper phage. Fluorescently labelled primer (P3) and structures annealing were performed before initiating DNA synthesis through the addition of T7 DNA polymerase. Primers are then either fully extended to the length of the circular template or the extension is stopped within STRs if the DNA polymerases stall at structured DNAs. (b) Extended and stalled products were then analysed by denaturing Poly Acrylamide Gel (PAGE) electrophoresis, recovered from the gel matrix and prepared for high throughput sequencing. DNA polymerase stalling was then quantified by analysing the enrichment of each sequence form the library in the stalled and extended fractions. Representative fluorescence gel imaging of primer extension reactions on templates containing a G-quadruplex (G4) structure, a mutated G4 or the entire DNA library, stopped after the indicated times, are reported for comparison. Blue and red arrows indicate the position of the extended and stalled products respectively. The green line highlights the presence of transient stall sites that disappear overtime.
    Figure Legend Snippet: Pooled measurement of DNA polymerase stalling at STRs. (a) Overview of the high-throughput primer extension assay used to monitor DNA synthesis at designed sequences. A library of 20,000 sequences comprising all STR permutations at three different lengths together with control structured DNA sequences was synthesised on a programmable microarray, eluted and inserted into a phagemid vector. After PCR amplification, insertion into a phagemid vector and bacterial amplification, circular single-stranded DNA templates were produced using a M13KO7 helper phage. Fluorescently labelled primer (P3) and structures annealing were performed before initiating DNA synthesis through the addition of T7 DNA polymerase. Primers are then either fully extended to the length of the circular template or the extension is stopped within STRs if the DNA polymerases stall at structured DNAs. (b) Extended and stalled products were then analysed by denaturing Poly Acrylamide Gel (PAGE) electrophoresis, recovered from the gel matrix and prepared for high throughput sequencing. DNA polymerase stalling was then quantified by analysing the enrichment of each sequence form the library in the stalled and extended fractions. Representative fluorescence gel imaging of primer extension reactions on templates containing a G-quadruplex (G4) structure, a mutated G4 or the entire DNA library, stopped after the indicated times, are reported for comparison. Blue and red arrows indicate the position of the extended and stalled products respectively. The green line highlights the presence of transient stall sites that disappear overtime.

    Techniques Used: High Throughput Screening Assay, Primer Extension Assay, DNA Synthesis, Microarray, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Produced, Acrylamide Gel Assay, Polyacrylamide Gel Electrophoresis, Electrophoresis, Next-Generation Sequencing, Sequencing, Fluorescence, Imaging

    29) Product Images from "DNA polymerase stalling at structured DNA constrains the expansion of Short Tandem Repeats"

    Article Title: DNA polymerase stalling at structured DNA constrains the expansion of Short Tandem Repeats

    Journal: bioRxiv

    doi: 10.1101/2020.06.20.162743

    Pooled measurement of DNA polymerase stalling at STRs. (a) Overview of the high-throughput primer extension assay used to monitor DNA synthesis at designed sequences. A library of 20,000 sequences comprising all STR permutations at three different lengths together with control structured DNA sequences was synthesised on a programmable microarray, eluted and inserted into a phagemid vector. After PCR amplification, insertion into a phagemid vector and bacterial amplification, circular single-stranded DNA templates were produced using a M13KO7 helper phage. Fluorescently labelled primer (P3) and structures annealing were performed before initiating DNA synthesis through the addition of T7 DNA polymerase. Primers are then either fully extended to the length of the circular template or the extension is stopped within STRs if the DNA polymerases stall at structured DNAs. (b) Extended and stalled products were then analysed by denaturing Poly Acrylamide Gel (PAGE) electrophoresis, recovered from the gel matrix and prepared for high throughput sequencing. DNA polymerase stalling was then quantified by analysing the enrichment of each sequence form the library in the stalled and extended fractions. Representative fluorescence gel imaging of primer extension reactions on templates containing a G-quadruplex (G4) structure, a mutated G4 or the entire DNA library, stopped after the indicated times, are reported for comparison. Blue and red arrows indicate the position of the extended and stalled products respectively. The green line highlights the presence of transient stall sites that disappear overtime.
    Figure Legend Snippet: Pooled measurement of DNA polymerase stalling at STRs. (a) Overview of the high-throughput primer extension assay used to monitor DNA synthesis at designed sequences. A library of 20,000 sequences comprising all STR permutations at three different lengths together with control structured DNA sequences was synthesised on a programmable microarray, eluted and inserted into a phagemid vector. After PCR amplification, insertion into a phagemid vector and bacterial amplification, circular single-stranded DNA templates were produced using a M13KO7 helper phage. Fluorescently labelled primer (P3) and structures annealing were performed before initiating DNA synthesis through the addition of T7 DNA polymerase. Primers are then either fully extended to the length of the circular template or the extension is stopped within STRs if the DNA polymerases stall at structured DNAs. (b) Extended and stalled products were then analysed by denaturing Poly Acrylamide Gel (PAGE) electrophoresis, recovered from the gel matrix and prepared for high throughput sequencing. DNA polymerase stalling was then quantified by analysing the enrichment of each sequence form the library in the stalled and extended fractions. Representative fluorescence gel imaging of primer extension reactions on templates containing a G-quadruplex (G4) structure, a mutated G4 or the entire DNA library, stopped after the indicated times, are reported for comparison. Blue and red arrows indicate the position of the extended and stalled products respectively. The green line highlights the presence of transient stall sites that disappear overtime.

    Techniques Used: High Throughput Screening Assay, Primer Extension Assay, DNA Synthesis, Microarray, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Produced, Acrylamide Gel Assay, Polyacrylamide Gel Electrophoresis, Electrophoresis, Next-Generation Sequencing, Sequencing, Fluorescence, Imaging

    30) Product Images from "Klebsiella Phage KP34 RNA Polymerase and Its Use in RNA Synthesis"

    Article Title: Klebsiella Phage KP34 RNA Polymerase and Its Use in RNA Synthesis

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2019.02487

    Identification of the KP34 RNAP promoter. (A) The DNA fragment (Template 1) containing previously predicated promoters failed to serve as transcription template for KP34 RNAP to produce RNA in vitro , while the DNA fragment covering the RNAP gene and downstream gap region (Template 2) was active as transcription template. RNA products are shown as bright bands on the 2% TAE agarose gel. (B) 5′-RACE analysis of the position of transcription initiation. 5′ sequence of KP34 transcripts were matched to KP34 genome. Major sequences were in solid box and minor sequences in dotted box. Their upstream region containing putative promoters is shown in bold. (C) Comparison of run-off RNA synthesis by T7 and KP34 RNAP under the control of various promoters. A DNA template containing a T7 promoter (5′-TAATACGACTCACTATA-3′) was incubated with 100 nM T7 RNAP, and three DNA templates containing either a KP34 strong promoter S1 (5′-TAATGTTACAGGAGTA-3′), a KP34 strong promoter S2 (5′-TGATGTTACAGGAGTA-3′), or a KP34 weak promoter W (5′-ACTTTGGACATCCG TCAAGT-3′) were incubated with 100 nM KP34 RNAP to direct to the transcription of their downstream sequence that encodes the same 37 nt RNA. [α-32P]ATP was added into reactions for imaging and visualization. Reaction products were separated by a 25% TBE-Urea denaturing gel. (D) Identification of the full KP34 strong promoter. A KP34 strong promoter S1 (5′-TAATGTTACAGGAGTA-3′) or the 3′ 14 nt common sequence of the two KP34 strong promoters (5′-ATGTTA CAGGAGTA-3′) was inserted into plasmid pUC19 to direct the transcription of their downstream sequences, respectively. A run-off transcript of ∼2700 nt and a terminated transcript of ∼1000 nt (terminated by a predictable T7 class I hairpin terminator structure) were expected from the linearized form of these plasmids if the inserted promoter is sufficient to direct transcription by KP34 RNAP. M: ssRNA Ladder. (E) KP34 promoters in the genome (location of strong promoter (S) pointed by solid arrow and weak promoter (W) by dotted arrow) and comparison of typical ssRNAP promoters. Conserved sequence among ssRNAP promoters are in bold and those homologous between Syn5 promoter and KP34 weak promoter are underlined.
    Figure Legend Snippet: Identification of the KP34 RNAP promoter. (A) The DNA fragment (Template 1) containing previously predicated promoters failed to serve as transcription template for KP34 RNAP to produce RNA in vitro , while the DNA fragment covering the RNAP gene and downstream gap region (Template 2) was active as transcription template. RNA products are shown as bright bands on the 2% TAE agarose gel. (B) 5′-RACE analysis of the position of transcription initiation. 5′ sequence of KP34 transcripts were matched to KP34 genome. Major sequences were in solid box and minor sequences in dotted box. Their upstream region containing putative promoters is shown in bold. (C) Comparison of run-off RNA synthesis by T7 and KP34 RNAP under the control of various promoters. A DNA template containing a T7 promoter (5′-TAATACGACTCACTATA-3′) was incubated with 100 nM T7 RNAP, and three DNA templates containing either a KP34 strong promoter S1 (5′-TAATGTTACAGGAGTA-3′), a KP34 strong promoter S2 (5′-TGATGTTACAGGAGTA-3′), or a KP34 weak promoter W (5′-ACTTTGGACATCCG TCAAGT-3′) were incubated with 100 nM KP34 RNAP to direct to the transcription of their downstream sequence that encodes the same 37 nt RNA. [α-32P]ATP was added into reactions for imaging and visualization. Reaction products were separated by a 25% TBE-Urea denaturing gel. (D) Identification of the full KP34 strong promoter. A KP34 strong promoter S1 (5′-TAATGTTACAGGAGTA-3′) or the 3′ 14 nt common sequence of the two KP34 strong promoters (5′-ATGTTA CAGGAGTA-3′) was inserted into plasmid pUC19 to direct the transcription of their downstream sequences, respectively. A run-off transcript of ∼2700 nt and a terminated transcript of ∼1000 nt (terminated by a predictable T7 class I hairpin terminator structure) were expected from the linearized form of these plasmids if the inserted promoter is sufficient to direct transcription by KP34 RNAP. M: ssRNA Ladder. (E) KP34 promoters in the genome (location of strong promoter (S) pointed by solid arrow and weak promoter (W) by dotted arrow) and comparison of typical ssRNAP promoters. Conserved sequence among ssRNAP promoters are in bold and those homologous between Syn5 promoter and KP34 weak promoter are underlined.

    Techniques Used: In Vitro, Agarose Gel Electrophoresis, Sequencing, Incubation, Imaging, Plasmid Preparation

    Synthesis of a 50 nt RNA containing 3′ hairpin structure by various RNAPs. (A) The 50 nt RNA sequence is shown at the top of the gel. The three DNA templates containing the same coding sequences for the 50 nt run-off RNA transcripts under the control of either a T7 promoter, a KP34 strong promoter, or a Syn5 promoter were incubated with 0.2 μM T7 RNAP, 1 μM KP34 RNAP, or 1 μM Syn5 RNAP, respectively. Incubation with KP34 and T7 RNAP was at 37°C for 1 h and incubation with Syn5 RNAP was at 24°C for 1 h. Reaction products were separated by a 12% TBE native gel and then stained with ethidium bromide. M: ssRNA Ladder. (B) RNA-Seq analysis of the 3′ termini of T7 RNAP transcripts. 3′ termini of sequences with reads more than 1% of total reads were aligned and shown. Percentage of major sequences in total sequencing results are noted and percentage of the correct product is in bold. A dotted line cut indicates the precise terminus encoded by DNA template, and the number of extended nt is shown as n + x. Bold sequences indicate complementary sequences in each RNA specie resulted from extension of a possible 3′ self-primed structure. (C) Similar as B, RNA-Seq analysis of the 3′ termini of KP34 RNAP transcripts. Number of missing nt at the 3′ terminus of major sequences is shown as n–x.
    Figure Legend Snippet: Synthesis of a 50 nt RNA containing 3′ hairpin structure by various RNAPs. (A) The 50 nt RNA sequence is shown at the top of the gel. The three DNA templates containing the same coding sequences for the 50 nt run-off RNA transcripts under the control of either a T7 promoter, a KP34 strong promoter, or a Syn5 promoter were incubated with 0.2 μM T7 RNAP, 1 μM KP34 RNAP, or 1 μM Syn5 RNAP, respectively. Incubation with KP34 and T7 RNAP was at 37°C for 1 h and incubation with Syn5 RNAP was at 24°C for 1 h. Reaction products were separated by a 12% TBE native gel and then stained with ethidium bromide. M: ssRNA Ladder. (B) RNA-Seq analysis of the 3′ termini of T7 RNAP transcripts. 3′ termini of sequences with reads more than 1% of total reads were aligned and shown. Percentage of major sequences in total sequencing results are noted and percentage of the correct product is in bold. A dotted line cut indicates the precise terminus encoded by DNA template, and the number of extended nt is shown as n + x. Bold sequences indicate complementary sequences in each RNA specie resulted from extension of a possible 3′ self-primed structure. (C) Similar as B, RNA-Seq analysis of the 3′ termini of KP34 RNAP transcripts. Number of missing nt at the 3′ terminus of major sequences is shown as n–x.

    Techniques Used: Sequencing, Incubation, Staining, RNA Sequencing Assay

    Synthesis of an sgRNA by T7 and KP34 RNAP. (A) The sgRNA sequence is shown at the top of the gel. The three DNA templates containing the same coding sequences for the sgRNA under the control of either a T7 promoter, a KP34 strong promoter, or a Syn5 promoter were incubated with 0.2 μM T7 RNAP, 1 μM KP34 RNAP, or 1 μM Syn5 RNAP, respectively. Incubation with KP34 and T7 RNAP was at 37°C for 1 h and incubation with Syn5 RNAP was at 24°C for 1 h. Reaction products were separated by a 12% TBE native gel and then stained with ethidium bromide. M: ssRNA Ladder. (B) 3′-RACE analysis of the 3′ termini of T7 RNAP transcripts. 3′ termini of obtained sequences were aligned and shown. A dotted line cut indicates the precise terminus encoded by DNA template and number of extended or missing nt is shown as n + x or n–x. Bold sequences indicate complementary sequences in each RNA resulted from extension of possible 3′ self-primed structures. (C) Similar as B, 3′-RACE analysis of the 3′ termini of KP34 RNAP transcripts.
    Figure Legend Snippet: Synthesis of an sgRNA by T7 and KP34 RNAP. (A) The sgRNA sequence is shown at the top of the gel. The three DNA templates containing the same coding sequences for the sgRNA under the control of either a T7 promoter, a KP34 strong promoter, or a Syn5 promoter were incubated with 0.2 μM T7 RNAP, 1 μM KP34 RNAP, or 1 μM Syn5 RNAP, respectively. Incubation with KP34 and T7 RNAP was at 37°C for 1 h and incubation with Syn5 RNAP was at 24°C for 1 h. Reaction products were separated by a 12% TBE native gel and then stained with ethidium bromide. M: ssRNA Ladder. (B) 3′-RACE analysis of the 3′ termini of T7 RNAP transcripts. 3′ termini of obtained sequences were aligned and shown. A dotted line cut indicates the precise terminus encoded by DNA template and number of extended or missing nt is shown as n + x or n–x. Bold sequences indicate complementary sequences in each RNA resulted from extension of possible 3′ self-primed structures. (C) Similar as B, 3′-RACE analysis of the 3′ termini of KP34 RNAP transcripts.

    Techniques Used: Sequencing, Incubation, Staining

    31) Product Images from "A selective class of inhibitors for the CLC-Ka chloride ion channel"

    Article Title: A selective class of inhibitors for the CLC-Ka chloride ion channel

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

    doi: 10.1073/pnas.1720584115

    Selectivity of BIM1 among mammalian CLC homologs. Representative currents from Xenopus oocytes expressing CLC-1 ( A ) or CLC-2 ( B ) shown before and after application of 100 µM BIM1. ( C ) Summary inhibition data (± SEM) for 100 µM BIM1 on CLC-1 ( n = 8), CLC-2 ( n = 8), CLC-Ka ( n = 9), and CLC-Kb ( n = 6). Inhibition is reported for data at +60 mV (CLC-Ka, CLC-Kb, and CLC-1) or −120 mV (CLC-2). For CLC-1 and CLC-2, inhibition is not significantly different from zero ( P = 0.55 and P = 0.84, respectively).
    Figure Legend Snippet: Selectivity of BIM1 among mammalian CLC homologs. Representative currents from Xenopus oocytes expressing CLC-1 ( A ) or CLC-2 ( B ) shown before and after application of 100 µM BIM1. ( C ) Summary inhibition data (± SEM) for 100 µM BIM1 on CLC-1 ( n = 8), CLC-2 ( n = 8), CLC-Ka ( n = 9), and CLC-Kb ( n = 6). Inhibition is reported for data at +60 mV (CLC-Ka, CLC-Kb, and CLC-1) or −120 mV (CLC-2). For CLC-1 and CLC-2, inhibition is not significantly different from zero ( P = 0.55 and P = 0.84, respectively).

    Techniques Used: Expressing, Inhibition

    32) Product Images from "AID–RNA polymerase II transcription-dependent deamination of IgV DNA"

    Article Title: AID–RNA polymerase II transcription-dependent deamination of IgV DNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkz821

    Experimental protocol used to analyze AID-catalyzed dC deamination on IGHV3-23*01 during transcription by human Pol II. ( A ) Pol II ± DSIF elongation complexes were assembled on a DNA–RNA ‘scaffolded bubble’ substrate and preincubated with AID. Transcription was initiated by the addition rNTP substrates, and the elongation reaction was performed at 30°C (Methods). Following transcription, Exonuclease I (Exo I) was added to digest ssDNA. TS and NTS DNAs were separately barcoded and subjected next-generation sequencing analysis using Maximum Depth Sequencing (MDS) ( 39 ) to assess AID-mediated dC deamination. ( B ) Transcription in the presence of AID and DSIF was visualized as 32 P-labeled RNA primer elongation bands that extend for the full length of the IgV DNA (198 nt). A strong transcription pause region is located ∼11 nt downstream of the scaffold bubble, and is followed by six C residues on the TS, in which deaminations are observed to occur at as many as three contiguous C sites – see also Supplemental Figure S9. ( C ) Distribution of Pol II extended transcripts. Percentage (mean ± standard deviation) of scaffold bubble proximal transcripts (1–12 nt from the end of the scaffold bubble to a run of six consecutive Cs) and full-length transcript (198 nt) were quantified by GE Healthcare ImageQuant software. A sketch depicting the transcribed IgV substrate and the scaffold bubble containing a 20 nt RNA primer strand is shown at the top.
    Figure Legend Snippet: Experimental protocol used to analyze AID-catalyzed dC deamination on IGHV3-23*01 during transcription by human Pol II. ( A ) Pol II ± DSIF elongation complexes were assembled on a DNA–RNA ‘scaffolded bubble’ substrate and preincubated with AID. Transcription was initiated by the addition rNTP substrates, and the elongation reaction was performed at 30°C (Methods). Following transcription, Exonuclease I (Exo I) was added to digest ssDNA. TS and NTS DNAs were separately barcoded and subjected next-generation sequencing analysis using Maximum Depth Sequencing (MDS) ( 39 ) to assess AID-mediated dC deamination. ( B ) Transcription in the presence of AID and DSIF was visualized as 32 P-labeled RNA primer elongation bands that extend for the full length of the IgV DNA (198 nt). A strong transcription pause region is located ∼11 nt downstream of the scaffold bubble, and is followed by six C residues on the TS, in which deaminations are observed to occur at as many as three contiguous C sites – see also Supplemental Figure S9. ( C ) Distribution of Pol II extended transcripts. Percentage (mean ± standard deviation) of scaffold bubble proximal transcripts (1–12 nt from the end of the scaffold bubble to a run of six consecutive Cs) and full-length transcript (198 nt) were quantified by GE Healthcare ImageQuant software. A sketch depicting the transcribed IgV substrate and the scaffold bubble containing a 20 nt RNA primer strand is shown at the top.

    Techniques Used: Next-Generation Sequencing, Sequencing, Labeling, Standard Deviation, Software

    A model for AID putative access to the NTS and TS during Pol II transcription. Sketch of how AID might interact with dC residues on both the TS or NTS to convert C → U. Pol II has been observed to pause and to backtrack during transcription elongation. We propose that paused or backtracked Pol II interacts with AID at the upstream edge of the transcription bubble where the TS and NTS strands exit the polymerase, as inferred from structural studies of Pol II elongation complexes. In this model, AID can interact with dC residues on both the TS or NTS to convert C → U. Structural and single-molecule resolution transcriptional data suggest that AID could have access to about a 15 nt region of transient ssDNA, corresponding to a stalled transcription bubble (∼10 nt) and perhaps an additional region of ssDNA resulting from a backtracked Pol II (∼5 nt).
    Figure Legend Snippet: A model for AID putative access to the NTS and TS during Pol II transcription. Sketch of how AID might interact with dC residues on both the TS or NTS to convert C → U. Pol II has been observed to pause and to backtrack during transcription elongation. We propose that paused or backtracked Pol II interacts with AID at the upstream edge of the transcription bubble where the TS and NTS strands exit the polymerase, as inferred from structural studies of Pol II elongation complexes. In this model, AID can interact with dC residues on both the TS or NTS to convert C → U. Structural and single-molecule resolution transcriptional data suggest that AID could have access to about a 15 nt region of transient ssDNA, corresponding to a stalled transcription bubble (∼10 nt) and perhaps an additional region of ssDNA resulting from a backtracked Pol II (∼5 nt).

    Techniques Used:

    33) Product Images from "Biochemically diverse CRISPR-Cas9 orthologs"

    Article Title: Biochemically diverse CRISPR-Cas9 orthologs

    Journal: bioRxiv

    doi: 10.1101/2020.04.29.066654

    Target DNA cleavage patterns produced by Cas9 orthologs. Cleavage sites and resultant dsDNA ends are depicted as heatmaps that show the proportion of cleaved ends recovered by DNA sequencing at each position of a target DNA. Intensity of the blue color indicates the proportion of mapped cleavage ends. (A) Control digests using restriction enzymes recover 5’-overhangs, 3’-overhangs, and blunt ends. TS indicates top strand; BS indicates bottom strand. (B) SpyCas9 and SauCas9 cleaved DNA ends. Heatmaps represent mapped cleavage ends as the averages at each position in 5 different dsDNA targets. Position of the DNA bases and PAM sequences is depicted above the heatmaps. NTS indicates non-target strand; TS indicates target strand. (C) Blunt and staggered-end cleavage. Examples of blunt, one base 5’-overhang staggered cleavage, and multiple base 5’-overhang cleavage are depicted as heatmaps that show the proportion of cleaved ends as the averages at each position in 5 different dsDNA targets. Position of the DNA bases and PAM sequences is depicted above the heatmaps. NTS indicates non-target strand; TS indicates target strand.
    Figure Legend Snippet: Target DNA cleavage patterns produced by Cas9 orthologs. Cleavage sites and resultant dsDNA ends are depicted as heatmaps that show the proportion of cleaved ends recovered by DNA sequencing at each position of a target DNA. Intensity of the blue color indicates the proportion of mapped cleavage ends. (A) Control digests using restriction enzymes recover 5’-overhangs, 3’-overhangs, and blunt ends. TS indicates top strand; BS indicates bottom strand. (B) SpyCas9 and SauCas9 cleaved DNA ends. Heatmaps represent mapped cleavage ends as the averages at each position in 5 different dsDNA targets. Position of the DNA bases and PAM sequences is depicted above the heatmaps. NTS indicates non-target strand; TS indicates target strand. (C) Blunt and staggered-end cleavage. Examples of blunt, one base 5’-overhang staggered cleavage, and multiple base 5’-overhang cleavage are depicted as heatmaps that show the proportion of cleaved ends as the averages at each position in 5 different dsDNA targets. Position of the DNA bases and PAM sequences is depicted above the heatmaps. NTS indicates non-target strand; TS indicates target strand.

    Techniques Used: Produced, DNA Sequencing

    Activity of Cas9 orthologs at varying temperatures. The cleavage activity of Cas9 orthologs was measured using in vitro DNA cleavage assays using fluorophore-labeled dsDNA substrates. Cleaved fragments were quantitated and are represented in a heat map (A) showing overall activity at temperatures ranging from 10°C to 68°C. (B) Cas9 orthologs with activity at elevated temperatures. In vitro DNA cleavage activity for a subset of Cas9 orthologs with > 50% activity at 53°C is summarized in a heat map and plotted as proportion of DNA substrate cleaved at varied temperature. Points represent the mean +/- SEM of at least 3 independent experiments. (C) Cas9 orthologs with reduced activity at room temperature. In vitro DNA cleavage activity for a subset of Cas9 orthologs with
    Figure Legend Snippet: Activity of Cas9 orthologs at varying temperatures. The cleavage activity of Cas9 orthologs was measured using in vitro DNA cleavage assays using fluorophore-labeled dsDNA substrates. Cleaved fragments were quantitated and are represented in a heat map (A) showing overall activity at temperatures ranging from 10°C to 68°C. (B) Cas9 orthologs with activity at elevated temperatures. In vitro DNA cleavage activity for a subset of Cas9 orthologs with > 50% activity at 53°C is summarized in a heat map and plotted as proportion of DNA substrate cleaved at varied temperature. Points represent the mean +/- SEM of at least 3 independent experiments. (C) Cas9 orthologs with reduced activity at room temperature. In vitro DNA cleavage activity for a subset of Cas9 orthologs with

    Techniques Used: Activity Assay, In Vitro, Labeling

    Cas9 PAM Interacting (PI) domain similarity. Cas9 PI domains clustered by their pairwise sequence similarity. Lines connect sequences with P-value ≤ 1e-11. Line shading corresponds to P-values according to the scale in the top-right corner (light and long lines connect distantly related sequences). Major clusters are shown in bold. Cluster 1 was so named to emphasize that it contains first experimentally characterized Cas9. Clusters 2 to 10 were named beginning from the one with the most members. Sequences having known structures are marked red, their PDB code is shown in parentheses.
    Figure Legend Snippet: Cas9 PAM Interacting (PI) domain similarity. Cas9 PI domains clustered by their pairwise sequence similarity. Lines connect sequences with P-value ≤ 1e-11. Line shading corresponds to P-values according to the scale in the top-right corner (light and long lines connect distantly related sequences). Major clusters are shown in bold. Cluster 1 was so named to emphasize that it contains first experimentally characterized Cas9. Clusters 2 to 10 were named beginning from the one with the most members. Sequences having known structures are marked red, their PDB code is shown in parentheses.

    Techniques Used: Sequencing

    Cas9 diversity and characterization approach. (A) Phylogenetic representation of the diversity provided by Cas9 orthologs. Type II-A, B, and C systems are color-coded, red, blue, and green, respectively. Distinct phylogenetic clades are numbered I-X. Those selected for study are indicated with a black dot. Cas9s whose structure has been determined are also designated. (B) Biochemical approach used to directly capture target cleavage and assess PAM recognition. Experiments were assembled using Cas9 protein produced by IVT.
    Figure Legend Snippet: Cas9 diversity and characterization approach. (A) Phylogenetic representation of the diversity provided by Cas9 orthologs. Type II-A, B, and C systems are color-coded, red, blue, and green, respectively. Distinct phylogenetic clades are numbered I-X. Those selected for study are indicated with a black dot. Cas9s whose structure has been determined are also designated. (B) Biochemical approach used to directly capture target cleavage and assess PAM recognition. Experiments were assembled using Cas9 protein produced by IVT.

    Techniques Used: Produced

    Cas9 tracrRNA sequence and secondary structure similarity. Circles are scaled based on the number of sequences belonging to each covariance model (CM) and colored according to the designated cluster. The width of the connecting lines indicates the percentage of similarity or relatedness among CMs. Representative tracrRNAs from each cluster are indicated with the associated color. CMs not assigned to a cluster are in grey.
    Figure Legend Snippet: Cas9 tracrRNA sequence and secondary structure similarity. Circles are scaled based on the number of sequences belonging to each covariance model (CM) and colored according to the designated cluster. The width of the connecting lines indicates the percentage of similarity or relatedness among CMs. Representative tracrRNAs from each cluster are indicated with the associated color. CMs not assigned to a cluster are in grey.

    Techniques Used: Sequencing

    34) Product Images from "Fluid flow-induced left-right asymmetric decay of Dand5 mRNA in the mouse embryo requires Bicc1-Ccr4 RNA degradation complex"

    Article Title: Fluid flow-induced left-right asymmetric decay of Dand5 mRNA in the mouse embryo requires Bicc1-Ccr4 RNA degradation complex

    Journal: bioRxiv

    doi: 10.1101/2020.02.02.931477

    Determination of Bicc1 Binding Motifs in RNA a Schematic representation of RNA Bind-n-Seq (RBNS), which determines RNA motifs enriched by target proteins with the use of a random RNA sequence library. 293FT cells were transfected with a plasmid for overexpression (O/E) of FLAG-tagged Bicc1. Cell lysates containing the Bicc1-FLAG protein were then mixed with a random RNA sequence library, and resulting RNA-protein complexes were isolated by immunoprecipitation with magnetic bead–conjugated antibodies to FLAG. Finally, the isolated RNA sequences were converted to a DNA library by RT-PCR for deep sequencing. b Analysis of the RBNS data set. The number of each k-mer (where k = 4, 5, or 6) RNA sequence was compared between cells transfected with the Bicc1-FLAG expression plasmid and those subjected to mock transfection (control). c A motif logo generated from aligned hexamers significantly enriched by Bicc1-FLAG. d Enriched 4-mer and 5-mer sequences sorted by relative frequency determined by comparison of the Bicc1-FLAG and control RBNS data. e Schematic representation of metagene analysis for the 200-nucleotide proximal region of the 3’-UTR of mouse mRNAs. A total of 31,16 5 regions extracted from mouse genes (mm10) was searched with the indicated target motifs. f Histogram of motif frequency revealed by metagene analysis. The vertical black and blue lines indicate the averaged frequency of each target motif and the frequency of each target motif in the 200-nucleotide proximal region of the 3’-UTR of Dand5 mRNA. g Maps of GAC-containing motifs in the 3’-UTR of Dand5 mRNAs for the indicated species.
    Figure Legend Snippet: Determination of Bicc1 Binding Motifs in RNA a Schematic representation of RNA Bind-n-Seq (RBNS), which determines RNA motifs enriched by target proteins with the use of a random RNA sequence library. 293FT cells were transfected with a plasmid for overexpression (O/E) of FLAG-tagged Bicc1. Cell lysates containing the Bicc1-FLAG protein were then mixed with a random RNA sequence library, and resulting RNA-protein complexes were isolated by immunoprecipitation with magnetic bead–conjugated antibodies to FLAG. Finally, the isolated RNA sequences were converted to a DNA library by RT-PCR for deep sequencing. b Analysis of the RBNS data set. The number of each k-mer (where k = 4, 5, or 6) RNA sequence was compared between cells transfected with the Bicc1-FLAG expression plasmid and those subjected to mock transfection (control). c A motif logo generated from aligned hexamers significantly enriched by Bicc1-FLAG. d Enriched 4-mer and 5-mer sequences sorted by relative frequency determined by comparison of the Bicc1-FLAG and control RBNS data. e Schematic representation of metagene analysis for the 200-nucleotide proximal region of the 3’-UTR of mouse mRNAs. A total of 31,16 5 regions extracted from mouse genes (mm10) was searched with the indicated target motifs. f Histogram of motif frequency revealed by metagene analysis. The vertical black and blue lines indicate the averaged frequency of each target motif and the frequency of each target motif in the 200-nucleotide proximal region of the 3’-UTR of Dand5 mRNA. g Maps of GAC-containing motifs in the 3’-UTR of Dand5 mRNAs for the indicated species.

    Techniques Used: Binding Assay, Sequencing, Transfection, Plasmid Preparation, Over Expression, Isolation, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Expressing, Generated

    35) Product Images from "Selection of an Efficient AAV Vector for Robust CNS Transgene Expression"

    Article Title: Selection of an Efficient AAV Vector for Robust CNS Transgene Expression

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1016/j.omtm.2019.10.007

    iTransduce Library for Selection of Novel AAV Capsids Capable of Efficient Transgene Expression in Target Tissue (A) Two-component system of the library construct. (1) Cre recombinase is driven by a minimal chicken β-actin (CBA) promoter. (2) p41 promoter-driven AAV9 capsid with random heptamer peptide is inserted between amino acids 588 and 589, cloned downstream of the Cre cassette. (B) Selection strategy. (Bi) The iTransduce library comprised of different peptide inserts expressed on the capsid (represented by different colors) is injected intravenously (i.v.) into an Ai9 transgenic mouse with a loxP-flanked STOP cassette upsteam of the tdTomato reporter gene, inserted into the Gt(ROSA)26Sor locus. AAV capsids able to enter the cell of interest but that do not functionally transduce the cell (no Cre expression) do not turn on tdTomato expression. Capsids that can mediate functional transduction (express Cre) will turn on tdTomato expression. (Bii) Cells are isolated from the organ of interest (e.g., brain), and transduced cells are sorted for tdTomato expression and optionally cell markers. (Biii) Capsid DNA is PCR amplified from the sorted cells, cloned back to the library vector, and repackaged for another round of selection. DNA sequencing analysis is utilized after each round to monitor the selection process.
    Figure Legend Snippet: iTransduce Library for Selection of Novel AAV Capsids Capable of Efficient Transgene Expression in Target Tissue (A) Two-component system of the library construct. (1) Cre recombinase is driven by a minimal chicken β-actin (CBA) promoter. (2) p41 promoter-driven AAV9 capsid with random heptamer peptide is inserted between amino acids 588 and 589, cloned downstream of the Cre cassette. (B) Selection strategy. (Bi) The iTransduce library comprised of different peptide inserts expressed on the capsid (represented by different colors) is injected intravenously (i.v.) into an Ai9 transgenic mouse with a loxP-flanked STOP cassette upsteam of the tdTomato reporter gene, inserted into the Gt(ROSA)26Sor locus. AAV capsids able to enter the cell of interest but that do not functionally transduce the cell (no Cre expression) do not turn on tdTomato expression. Capsids that can mediate functional transduction (express Cre) will turn on tdTomato expression. (Bii) Cells are isolated from the organ of interest (e.g., brain), and transduced cells are sorted for tdTomato expression and optionally cell markers. (Biii) Capsid DNA is PCR amplified from the sorted cells, cloned back to the library vector, and repackaged for another round of selection. DNA sequencing analysis is utilized after each round to monitor the selection process.

    Techniques Used: Selection, Expressing, Construct, Crocin Bleaching Assay, Clone Assay, Injection, Transgenic Assay, Functional Assay, Transduction, Isolation, Polymerase Chain Reaction, Amplification, Plasmid Preparation, DNA Sequencing

    36) Product Images from "Stochastic transcription in the p53‐mediated response to DNA damage is modulated by burst frequency"

    Article Title: Stochastic transcription in the p53‐mediated response to DNA damage is modulated by burst frequency

    Journal: Molecular Systems Biology

    doi: 10.15252/msb.20199068

    Single‐cell quantification of RNA expression by sm FISH highlights strong heterogeneity of p53 target gene expression p53 has been shown to response with a series of undamped pulse to ionizing irradiation leading to cell cycle arrest while intrinsic DNA damage during cell cycle does not induce regular pulsatile p53 and subsequent gene expression programs. Schematic representations of p53 dynamics in both cellular conditions are shown. We selected p53 target genes that are involved in different cell fate programs ranging from apoptosis (BAX), DNA repair (DDB2) cell cycle arrest (CDKN1A), proliferation control (SESN1), and the regulation of the p53 network itself (PPM1D and MDM2). Induction of selected p53 target genes after DNA damage induction in A549 wild‐type and p53 knockdown cells. RNA levels were measured by qRT–PCR before and 3 h after treatment with 10 Gy IR. Fold changes relative to basal levels are shown for each cell line as mean and standard deviation from technical triplicates. Fluorescence microscopy images of smFISH probes labeled with CAL Fluor 610 (gray) overlayed with Hoechst 33342 stainings (blue) for the indicated target genes in untreated A549 cells. Scale bar corresponds to 10 μm distance; images were contrast‐ and brightness‐enhanced for better visualization. Histograms of quantitative analysis of RNAs per cell for each target gene in the absence of DNA damage (basal). smFISH staining and quantitative analysis of p53 targets show broad variability of RNA counts per cell for all genes in basal conditions. Dashed line: median; solid line: probability density estimate (see Data visualization section), CV: coefficient of variation, Fano: Fano factor, m: median, n : number of cells analyzed. Source data are available online for this figure.
    Figure Legend Snippet: Single‐cell quantification of RNA expression by sm FISH highlights strong heterogeneity of p53 target gene expression p53 has been shown to response with a series of undamped pulse to ionizing irradiation leading to cell cycle arrest while intrinsic DNA damage during cell cycle does not induce regular pulsatile p53 and subsequent gene expression programs. Schematic representations of p53 dynamics in both cellular conditions are shown. We selected p53 target genes that are involved in different cell fate programs ranging from apoptosis (BAX), DNA repair (DDB2) cell cycle arrest (CDKN1A), proliferation control (SESN1), and the regulation of the p53 network itself (PPM1D and MDM2). Induction of selected p53 target genes after DNA damage induction in A549 wild‐type and p53 knockdown cells. RNA levels were measured by qRT–PCR before and 3 h after treatment with 10 Gy IR. Fold changes relative to basal levels are shown for each cell line as mean and standard deviation from technical triplicates. Fluorescence microscopy images of smFISH probes labeled with CAL Fluor 610 (gray) overlayed with Hoechst 33342 stainings (blue) for the indicated target genes in untreated A549 cells. Scale bar corresponds to 10 μm distance; images were contrast‐ and brightness‐enhanced for better visualization. Histograms of quantitative analysis of RNAs per cell for each target gene in the absence of DNA damage (basal). smFISH staining and quantitative analysis of p53 targets show broad variability of RNA counts per cell for all genes in basal conditions. Dashed line: median; solid line: probability density estimate (see Data visualization section), CV: coefficient of variation, Fano: Fano factor, m: median, n : number of cells analyzed. Source data are available online for this figure.

    Techniques Used: RNA Expression, Fluorescence In Situ Hybridization, Expressing, Irradiation, Quantitative RT-PCR, Standard Deviation, Fluorescence, Microscopy, Labeling, Staining

    Smyd2 and Set8 activities affect p53 nuclear dynamics and promoter binding Western blot of acetylated p53 (K370/K382) in A549 Smyd2 and Set8 knockdown cells compared to wild‐type cell lines shows an increase in acetylation specifically at later time points in the DNA damage response. Dynamics of total p53 remained pulse like. GAPDH is shown as loading control. Amount of p53 bound to CDKN1A and MDM2 promoters in A549 Smyd2 (B) and Set8 (C) knockdown cells before (basal, gray) and 3 h (red), 6 h (blue), and 9 h (orange) after DNA damage (10 Gy IR) as measured by ChIP. The amount of bound p53 was calculated as percentage of input and normalized to the time point of the first p53 peak at 3 h. Individual data points (mean values of triplicate quantification in qRT–PCR measurements) from two biological repeats are shown as dots; mean values are displayed as black horizontal lines. Dashed lines serve as guide to the eyes. We observed an increase in promoter binding at later time points similar to the results after Nutlin‐3 treatment.
    Figure Legend Snippet: Smyd2 and Set8 activities affect p53 nuclear dynamics and promoter binding Western blot of acetylated p53 (K370/K382) in A549 Smyd2 and Set8 knockdown cells compared to wild‐type cell lines shows an increase in acetylation specifically at later time points in the DNA damage response. Dynamics of total p53 remained pulse like. GAPDH is shown as loading control. Amount of p53 bound to CDKN1A and MDM2 promoters in A549 Smyd2 (B) and Set8 (C) knockdown cells before (basal, gray) and 3 h (red), 6 h (blue), and 9 h (orange) after DNA damage (10 Gy IR) as measured by ChIP. The amount of bound p53 was calculated as percentage of input and normalized to the time point of the first p53 peak at 3 h. Individual data points (mean values of triplicate quantification in qRT–PCR measurements) from two biological repeats are shown as dots; mean values are displayed as black horizontal lines. Dashed lines serve as guide to the eyes. We observed an increase in promoter binding at later time points similar to the results after Nutlin‐3 treatment.

    Techniques Used: Binding Assay, Western Blot, Chromatin Immunoprecipitation, Quantitative RT-PCR

    Sm FISH ‐based analysis at the first and second p53 pulse after IR reveals gene‐specific stochastic expression patterns Schematic illustration of the life cycle of an mRNA and the rate constants that influence RNA abundance due to stochastic bursting according to previously published models of promoter activity. While burst frequency (bf) describes the switching of a promoter between a transcriptionally active and inactive state with the rate constants k on and k off, the burst size (bs) describes the number of RNAs transcribed in an active period. Additionally, degradation (δ) further influences RNA levels by reducing the cytoplasmic RNA pool. Illustration of promoter activity according to the random telegraph model. An increase in RNA levels per cell can be due to a higher burst frequency (more active promoter periods, a higher rate of transcription initiation), or an increase in burst size (a higher rate of RNA transcription in an active period). Additionally, also mixtures of both scenarios are possible. We used smFISH data to calculated promoter activity based on previously published models. An overview of the calculations characterizing stochastic gene expression is shown. X RNA : number of quantified RNAs/cell, n : number of genomic loci, f : fraction of active promoters (proxy for burst frequency bf), μ: transcription rate per cell [RNA/h] (proxy for burst size bs), δ RNA : RNA degradation rate per cell [1/h], M : polymerase occupancy [RNAs/h], v : RNAP2 speed (estimated as 3 kb/min), l : gene length, TSS: active TSS at the moment of measurement. Further details can be found in Materials and Methods section. Quantification of stochastic gene expression for the indicated p53 target genes before (basal, gray) and 3 h (red), 6 h (blue), and 9 h (orange) after DNA damage (10 Gy IR). The fraction (f) of active promoters (proxy for burst frequency) increases, while the transcription rate (μ; proxy for burst size) at active TSS remains similar upon DNA damage for all time points. Left panel: The percentage of cells with active TSS is shown as stacked bar graphs. We subdivided the population in cells with strong TSS activity ( > 75% of TSS active, solid colors) and those with partial TSS activity (at least one, but less than 75% of TSS active, shaded colors). The mean fraction of active promoters (ratio of all active TSS to the total number of genomic loci analyzed) is indicated above each bar. Right panel: Distributions of calculated transcription rates μ [RNAs/h] at active TSS are presented for each time point as probability density estimates (PDF, see Data Visualization section). The number of TSS analyzed is indicated in each plot (compare Fig EV2 C). Mean degradation rates of indicated RNAs in transcriptionally active cells before (basal, gray) and 3 h (red), 6 h (blue), and 9 h (orange) after DNA damage (10 Gy IR) as calculated from smFISH data. RNA stability is not changing in the measured time frame upon DNA damage. The plot displays the average RNA degradation rate per cell [1/h] over time after DNA damage, calculated from model (C) in actively transcribing cells for each gene. Based on promoter activity, we allocated target gene promoters along three archetypical expression patterns illustrated by a schematic triangle. Amount of p53 bound to indicated target gene promoters before (basal, gray) and 3 h (red), 6 h (blue), and 9 h (orange) after DNA damage (10 Gy IR) as measured by ChIP. The amount of bound p53 was calculated as percentage of input and normalized to the time point of the first p53 peak at 3 h. Individual data points (mean values of triplicate quantification in qRT–PCR measurements) from 3 to 4 biological repeats are shown as dots; mean values are displayed as black horizontal lines. Dashed lines serve as guide to the eyes. We could not detect p53 binding above IgG controls at the published p53 response element in the PPM1D promoter (indicated by n.d.) Source data are available online for this figure.
    Figure Legend Snippet: Sm FISH ‐based analysis at the first and second p53 pulse after IR reveals gene‐specific stochastic expression patterns Schematic illustration of the life cycle of an mRNA and the rate constants that influence RNA abundance due to stochastic bursting according to previously published models of promoter activity. While burst frequency (bf) describes the switching of a promoter between a transcriptionally active and inactive state with the rate constants k on and k off, the burst size (bs) describes the number of RNAs transcribed in an active period. Additionally, degradation (δ) further influences RNA levels by reducing the cytoplasmic RNA pool. Illustration of promoter activity according to the random telegraph model. An increase in RNA levels per cell can be due to a higher burst frequency (more active promoter periods, a higher rate of transcription initiation), or an increase in burst size (a higher rate of RNA transcription in an active period). Additionally, also mixtures of both scenarios are possible. We used smFISH data to calculated promoter activity based on previously published models. An overview of the calculations characterizing stochastic gene expression is shown. X RNA : number of quantified RNAs/cell, n : number of genomic loci, f : fraction of active promoters (proxy for burst frequency bf), μ: transcription rate per cell [RNA/h] (proxy for burst size bs), δ RNA : RNA degradation rate per cell [1/h], M : polymerase occupancy [RNAs/h], v : RNAP2 speed (estimated as 3 kb/min), l : gene length, TSS: active TSS at the moment of measurement. Further details can be found in Materials and Methods section. Quantification of stochastic gene expression for the indicated p53 target genes before (basal, gray) and 3 h (red), 6 h (blue), and 9 h (orange) after DNA damage (10 Gy IR). The fraction (f) of active promoters (proxy for burst frequency) increases, while the transcription rate (μ; proxy for burst size) at active TSS remains similar upon DNA damage for all time points. Left panel: The percentage of cells with active TSS is shown as stacked bar graphs. We subdivided the population in cells with strong TSS activity ( > 75% of TSS active, solid colors) and those with partial TSS activity (at least one, but less than 75% of TSS active, shaded colors). The mean fraction of active promoters (ratio of all active TSS to the total number of genomic loci analyzed) is indicated above each bar. Right panel: Distributions of calculated transcription rates μ [RNAs/h] at active TSS are presented for each time point as probability density estimates (PDF, see Data Visualization section). The number of TSS analyzed is indicated in each plot (compare Fig EV2 C). Mean degradation rates of indicated RNAs in transcriptionally active cells before (basal, gray) and 3 h (red), 6 h (blue), and 9 h (orange) after DNA damage (10 Gy IR) as calculated from smFISH data. RNA stability is not changing in the measured time frame upon DNA damage. The plot displays the average RNA degradation rate per cell [1/h] over time after DNA damage, calculated from model (C) in actively transcribing cells for each gene. Based on promoter activity, we allocated target gene promoters along three archetypical expression patterns illustrated by a schematic triangle. Amount of p53 bound to indicated target gene promoters before (basal, gray) and 3 h (red), 6 h (blue), and 9 h (orange) after DNA damage (10 Gy IR) as measured by ChIP. The amount of bound p53 was calculated as percentage of input and normalized to the time point of the first p53 peak at 3 h. Individual data points (mean values of triplicate quantification in qRT–PCR measurements) from 3 to 4 biological repeats are shown as dots; mean values are displayed as black horizontal lines. Dashed lines serve as guide to the eyes. We could not detect p53 binding above IgG controls at the published p53 response element in the PPM1D promoter (indicated by n.d.) Source data are available online for this figure.

    Techniques Used: Fluorescence In Situ Hybridization, Expressing, Activity Assay, Chromatin Immunoprecipitation, Quantitative RT-PCR, Binding Assay

    The interplay of p53's C‐terminal lysine acetylation and methylation regulates transiently expressed target genes in response to IR A schematic illustration of p53's C‐terminal modifications and described functional implications, including key regulatory enzymes. Total p53, p53 acetylated at K382 and K370 as well as GAPDH were measured by Western blot at indicated time points in the context of different p53 dynamics: pulsing p53 (10 Gy IR), transient p53 (10 Gy IR + BML‐277, central lanes), and sustained p53 (10 Gy IR + Nutlin‐3, right lanes). See Fig 3 and Materials and Methods section for details. The relative change in p53 acetylation at K370 (light green) and K382 (dark green) was quantified from Western blot and normalized to the abundance 3 h post‐IR. Means and propagated standard errors from three independent experiments are indicated. Acetylation increased over time in the context of sustained p53. See also Appendix Fig S12 . The p53‐K370 methylase Smyd2 was down‐regulated in a clonal stable A549 cell line expressing a corresponding shRNA. Transcript levels were measured in wild‐type and knockdown cells by qRT–PCR. Mean levels and standard deviation from technical triplicates are indicated. Promoter activity of CDKN1A (E) and MDM2 (F) was quantified in Smyd2 knockdown cells before (basal, gray) and 3 h (red), 6 h (blue), and 9 h (orange) after DNA damage (10 Gy IR). Left panel: The percentage of cells with active TSS, subdivided into populations with strong ( > 75% of TSS, solid colors) and weak (
    Figure Legend Snippet: The interplay of p53's C‐terminal lysine acetylation and methylation regulates transiently expressed target genes in response to IR A schematic illustration of p53's C‐terminal modifications and described functional implications, including key regulatory enzymes. Total p53, p53 acetylated at K382 and K370 as well as GAPDH were measured by Western blot at indicated time points in the context of different p53 dynamics: pulsing p53 (10 Gy IR), transient p53 (10 Gy IR + BML‐277, central lanes), and sustained p53 (10 Gy IR + Nutlin‐3, right lanes). See Fig 3 and Materials and Methods section for details. The relative change in p53 acetylation at K370 (light green) and K382 (dark green) was quantified from Western blot and normalized to the abundance 3 h post‐IR. Means and propagated standard errors from three independent experiments are indicated. Acetylation increased over time in the context of sustained p53. See also Appendix Fig S12 . The p53‐K370 methylase Smyd2 was down‐regulated in a clonal stable A549 cell line expressing a corresponding shRNA. Transcript levels were measured in wild‐type and knockdown cells by qRT–PCR. Mean levels and standard deviation from technical triplicates are indicated. Promoter activity of CDKN1A (E) and MDM2 (F) was quantified in Smyd2 knockdown cells before (basal, gray) and 3 h (red), 6 h (blue), and 9 h (orange) after DNA damage (10 Gy IR). Left panel: The percentage of cells with active TSS, subdivided into populations with strong ( > 75% of TSS, solid colors) and weak (

    Techniques Used: Methylation, Functional Assay, Western Blot, Expressing, shRNA, Quantitative RT-PCR, Standard Deviation, Activity Assay

    37) Product Images from "Bacterial defenses against a natural antibiotic promote collateral resilience to clinical antibiotics"

    Article Title: Bacterial defenses against a natural antibiotic promote collateral resilience to clinical antibiotics

    Journal: bioRxiv

    doi: 10.1101/2020.04.20.049437

    PYO induces expression of specific efflux systems, conferring cross-tolerance to fluoroquinolones. A . Structures of PYO, two representative fluoroquinolones (CP = ciprofloxacin, LV = levofloxacin) and two representative aminoglycosides (GM = gentamicin, TM = tobramycin). PYO and fluoroquinolones are pumped by MexEF-OprN and MexGHI-OpmD, while aminoglycosides are not 11 . Rings with an aromatic character are highlighted in red. B . Normalized cDNA levels for genes within operons coding for the 11 main RND efflux systems in P. aeruginosa (left). PYO-dose-dependent changes in expression of mexEF-oprN and mexGHI- opmD systems (right; n = 3). For full qRT-PCR dataset, see Figs. S2, S3 and S4. C . Effect of PYO on tolerance to CP and LV in glucose minimal medium (left), and to CP in SCFM (right) (all 1 µg/mL) (n = 4). PYO itself was not toxic under the experimental conditions 8 . WT made 50-80 µM PYO as measured by absorbance of the culture supernatant at 691 nm. See Fig. S5A for experimental design. D-E . Effect of PYO on lag during outgrowth after exposure to CP. A representative field of view over different time points (D; magenta = WT::mApple, green = Δ phz ::GFP; see Movie S1) is shown together with the quantification of growth area on the agarose pads at time 0 hrs and 15 hrs (E). For these experiments, a culture of each strain tested was grown and exposed to CP (10 µg/mL) separately, then cells of both cultures were washed, mixed and placed together on a pad and imaged during outgrowth. The pads did not contain any PYO or CP (see Methods and Fig. S5D for details). White arrows in the displayed images point to regions with faster recovery of WT growth. The field of view displayed is marked with a black arrow in the quantification plot. The results for the experiment with swapped fluorescent proteins are shown in Fig. S5E. Scale bar: 20 µm. F . Tolerance of Δ phz to CP (1 µg/mL) in stationary phase in the presence of different concentrations of PYO (n = 4). G . Tolerance of Δ phz to CP (1 µg/mL) upon artificial induction of the mexGHI-opmD operon with arabinose (n = 4). The dashed green line marks the average survival of PYO-producing WT under similar conditions (without arabinose). Statistics: C, F – One-way ANOVA with Tukey’s HSD multiple-comparison test, with asterisks showing significant differences relative to untreated Δ phz (no PYO); E, G – Welch’s unpaired t- test (* p
    Figure Legend Snippet: PYO induces expression of specific efflux systems, conferring cross-tolerance to fluoroquinolones. A . Structures of PYO, two representative fluoroquinolones (CP = ciprofloxacin, LV = levofloxacin) and two representative aminoglycosides (GM = gentamicin, TM = tobramycin). PYO and fluoroquinolones are pumped by MexEF-OprN and MexGHI-OpmD, while aminoglycosides are not 11 . Rings with an aromatic character are highlighted in red. B . Normalized cDNA levels for genes within operons coding for the 11 main RND efflux systems in P. aeruginosa (left). PYO-dose-dependent changes in expression of mexEF-oprN and mexGHI- opmD systems (right; n = 3). For full qRT-PCR dataset, see Figs. S2, S3 and S4. C . Effect of PYO on tolerance to CP and LV in glucose minimal medium (left), and to CP in SCFM (right) (all 1 µg/mL) (n = 4). PYO itself was not toxic under the experimental conditions 8 . WT made 50-80 µM PYO as measured by absorbance of the culture supernatant at 691 nm. See Fig. S5A for experimental design. D-E . Effect of PYO on lag during outgrowth after exposure to CP. A representative field of view over different time points (D; magenta = WT::mApple, green = Δ phz ::GFP; see Movie S1) is shown together with the quantification of growth area on the agarose pads at time 0 hrs and 15 hrs (E). For these experiments, a culture of each strain tested was grown and exposed to CP (10 µg/mL) separately, then cells of both cultures were washed, mixed and placed together on a pad and imaged during outgrowth. The pads did not contain any PYO or CP (see Methods and Fig. S5D for details). White arrows in the displayed images point to regions with faster recovery of WT growth. The field of view displayed is marked with a black arrow in the quantification plot. The results for the experiment with swapped fluorescent proteins are shown in Fig. S5E. Scale bar: 20 µm. F . Tolerance of Δ phz to CP (1 µg/mL) in stationary phase in the presence of different concentrations of PYO (n = 4). G . Tolerance of Δ phz to CP (1 µg/mL) upon artificial induction of the mexGHI-opmD operon with arabinose (n = 4). The dashed green line marks the average survival of PYO-producing WT under similar conditions (without arabinose). Statistics: C, F – One-way ANOVA with Tukey’s HSD multiple-comparison test, with asterisks showing significant differences relative to untreated Δ phz (no PYO); E, G – Welch’s unpaired t- test (* p

    Techniques Used: Expressing, Quantitative RT-PCR

    38) Product Images from "DNA polymerase stalling at structured DNA constrains the expansion of short tandem repeats"

    Article Title: DNA polymerase stalling at structured DNA constrains the expansion of short tandem repeats

    Journal: Genome Biology

    doi: 10.1186/s13059-020-02124-x

    Pooled measurement of DNA polymerase stalling at STRs. a Overview of the high-throughput primer extension assay used to monitor DNA synthesis at designed sequences. A library of 20,000 sequences comprising all STR permutations at three different lengths together with control structured DNA sequences was synthesised on a programmable microarray, eluted and inserted into a phagemid vector. After PCR amplification, insertion into a phagemid vector and bacterial amplification, circular single-stranded DNA templates were produced using a M13KO7 helper phage. Fluorescently labelled primer (P3) and structure annealing were performed before initiating DNA synthesis through the addition of T7 DNA polymerase. Primers are then either fully extended to the length of the circular template, or the extension is stopped within STRs if the DNA polymerases stall at structured DNAs. b Extended and stalled products were then analysed by denaturing poly acrylamide gel electrophoresis (PAGE), recovered from the gel matrix and prepared for high-throughput sequencing. DNA polymerase stalling was then quantified by analysing the enrichment of each sequence from the library in the stalled and extended fractions. Representative fluorescence gel imaging of primer extension reactions on templates containing a G-quadruplex (G4) structure, a mutated G4 or the entire DNA library, stopped after the indicated times, is reported for comparison. Blue and red arrows indicate the position of the extended and stalled products respectively. The green line highlights the presence of transient stall sites that disappear over time
    Figure Legend Snippet: Pooled measurement of DNA polymerase stalling at STRs. a Overview of the high-throughput primer extension assay used to monitor DNA synthesis at designed sequences. A library of 20,000 sequences comprising all STR permutations at three different lengths together with control structured DNA sequences was synthesised on a programmable microarray, eluted and inserted into a phagemid vector. After PCR amplification, insertion into a phagemid vector and bacterial amplification, circular single-stranded DNA templates were produced using a M13KO7 helper phage. Fluorescently labelled primer (P3) and structure annealing were performed before initiating DNA synthesis through the addition of T7 DNA polymerase. Primers are then either fully extended to the length of the circular template, or the extension is stopped within STRs if the DNA polymerases stall at structured DNAs. b Extended and stalled products were then analysed by denaturing poly acrylamide gel electrophoresis (PAGE), recovered from the gel matrix and prepared for high-throughput sequencing. DNA polymerase stalling was then quantified by analysing the enrichment of each sequence from the library in the stalled and extended fractions. Representative fluorescence gel imaging of primer extension reactions on templates containing a G-quadruplex (G4) structure, a mutated G4 or the entire DNA library, stopped after the indicated times, is reported for comparison. Blue and red arrows indicate the position of the extended and stalled products respectively. The green line highlights the presence of transient stall sites that disappear over time

    Techniques Used: High Throughput Screening Assay, Primer Extension Assay, DNA Synthesis, Microarray, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Produced, Polyacrylamide Gel Electrophoresis, Next-Generation Sequencing, Sequencing, Fluorescence, Imaging

    39) Product Images from "DNA polymerase stalling at structured DNA constrains the expansion of short tandem repeats"

    Article Title: DNA polymerase stalling at structured DNA constrains the expansion of short tandem repeats

    Journal: Genome Biology

    doi: 10.1186/s13059-020-02124-x

    Pooled measurement of DNA polymerase stalling at STRs. a Overview of the high-throughput primer extension assay used to monitor DNA synthesis at designed sequences. A library of 20,000 sequences comprising all STR permutations at three different lengths together with control structured DNA sequences was synthesised on a programmable microarray, eluted and inserted into a phagemid vector. After PCR amplification, insertion into a phagemid vector and bacterial amplification, circular single-stranded DNA templates were produced using a M13KO7 helper phage. Fluorescently labelled primer (P3) and structure annealing were performed before initiating DNA synthesis through the addition of T7 DNA polymerase. Primers are then either fully extended to the length of the circular template, or the extension is stopped within STRs if the DNA polymerases stall at structured DNAs. b Extended and stalled products were then analysed by denaturing poly acrylamide gel electrophoresis (PAGE), recovered from the gel matrix and prepared for high-throughput sequencing. DNA polymerase stalling was then quantified by analysing the enrichment of each sequence from the library in the stalled and extended fractions. Representative fluorescence gel imaging of primer extension reactions on templates containing a G-quadruplex (G4) structure, a mutated G4 or the entire DNA library, stopped after the indicated times, is reported for comparison. Blue and red arrows indicate the position of the extended and stalled products respectively. The green line highlights the presence of transient stall sites that disappear over time
    Figure Legend Snippet: Pooled measurement of DNA polymerase stalling at STRs. a Overview of the high-throughput primer extension assay used to monitor DNA synthesis at designed sequences. A library of 20,000 sequences comprising all STR permutations at three different lengths together with control structured DNA sequences was synthesised on a programmable microarray, eluted and inserted into a phagemid vector. After PCR amplification, insertion into a phagemid vector and bacterial amplification, circular single-stranded DNA templates were produced using a M13KO7 helper phage. Fluorescently labelled primer (P3) and structure annealing were performed before initiating DNA synthesis through the addition of T7 DNA polymerase. Primers are then either fully extended to the length of the circular template, or the extension is stopped within STRs if the DNA polymerases stall at structured DNAs. b Extended and stalled products were then analysed by denaturing poly acrylamide gel electrophoresis (PAGE), recovered from the gel matrix and prepared for high-throughput sequencing. DNA polymerase stalling was then quantified by analysing the enrichment of each sequence from the library in the stalled and extended fractions. Representative fluorescence gel imaging of primer extension reactions on templates containing a G-quadruplex (G4) structure, a mutated G4 or the entire DNA library, stopped after the indicated times, is reported for comparison. Blue and red arrows indicate the position of the extended and stalled products respectively. The green line highlights the presence of transient stall sites that disappear over time

    Techniques Used: High Throughput Screening Assay, Primer Extension Assay, DNA Synthesis, Microarray, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Produced, Polyacrylamide Gel Electrophoresis, Next-Generation Sequencing, Sequencing, Fluorescence, Imaging

    40) Product Images from "Hermaphroditism in Marijuana (Cannabis sativa L.) Inflorescences – Impact on Floral Morphology, Seed Formation, Progeny Sex Ratios, and Genetic Variation"

    Article Title: Hermaphroditism in Marijuana (Cannabis sativa L.) Inflorescences – Impact on Floral Morphology, Seed Formation, Progeny Sex Ratios, and Genetic Variation

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2020.00718

    Sequence alignment of PCR fragments from female and male Cannabis sativa plants corresponding to the 540 bp band in female strains (F), the 540 bp band in male strains (M-L) and the 390 bp band in male strains (M-s). Only overlapping sequences were aligned for comparison. (A) Female sequence alignment showed 89.4–100% identity. The “*” designates sequences which have been submitted to NCBI with Accession Nos. MK093855 , MK093861 , and MK093858 , respectively. (B) Male sequence ali gnment showed 95.1–97% identity in M-L and 97.5–100% sequence identity in M-s. There are 2 regions of DNA present in the 540 bp bands that are absent in the 390 bp male bands, indicating indels in this region. All 390 bp male sequences share this deleted region. The “*” designates sequences which have been submitted to NCBI with Accession nos. MK093856 , MK093859 , MK093854 , MK093857 , MK093862 , and MK093860 , respectively.
    Figure Legend Snippet: Sequence alignment of PCR fragments from female and male Cannabis sativa plants corresponding to the 540 bp band in female strains (F), the 540 bp band in male strains (M-L) and the 390 bp band in male strains (M-s). Only overlapping sequences were aligned for comparison. (A) Female sequence alignment showed 89.4–100% identity. The “*” designates sequences which have been submitted to NCBI with Accession Nos. MK093855 , MK093861 , and MK093858 , respectively. (B) Male sequence ali gnment showed 95.1–97% identity in M-L and 97.5–100% sequence identity in M-s. There are 2 regions of DNA present in the 540 bp bands that are absent in the 390 bp male bands, indicating indels in this region. All 390 bp male sequences share this deleted region. The “*” designates sequences which have been submitted to NCBI with Accession nos. MK093856 , MK093859 , MK093854 , MK093857 , MK093862 , and MK093860 , respectively.

    Techniques Used: Sequencing, Polymerase Chain Reaction

    Conserved sequence domains present in representative female and male Cannabis sativa plants. (A) Conserved domains present in a female plant of strain “Space Queen” (SPQ) (Accession no. MK093858 ). Primers S22645strt and S22645end were used to amplify this region in the female genome beyond the ∼540 bp band produced by the GreenScreen primers. The size of the fragment, named SPQ(F)FL, is 1,190 bp. (B) Genious 11.1.5 pairwise alignment of representative male sequences (540 and 390 bp sizes) from strain “Jarilla.” The two indel regions total ∼178 bp. The 540 bp sequence (M-L) has the rve Superfamily pfam00665 whereas the 390 bp sequence (M-s) has the rve Superfamily core domain cl21549. (C) Structure of Copia and Gypsy LTR retrotransposons. This image was taken from the GyDB Gypsy Database 2.0 ( http://gydb.org ).
    Figure Legend Snippet: Conserved sequence domains present in representative female and male Cannabis sativa plants. (A) Conserved domains present in a female plant of strain “Space Queen” (SPQ) (Accession no. MK093858 ). Primers S22645strt and S22645end were used to amplify this region in the female genome beyond the ∼540 bp band produced by the GreenScreen primers. The size of the fragment, named SPQ(F)FL, is 1,190 bp. (B) Genious 11.1.5 pairwise alignment of representative male sequences (540 and 390 bp sizes) from strain “Jarilla.” The two indel regions total ∼178 bp. The 540 bp sequence (M-L) has the rve Superfamily pfam00665 whereas the 390 bp sequence (M-s) has the rve Superfamily core domain cl21549. (C) Structure of Copia and Gypsy LTR retrotransposons. This image was taken from the GyDB Gypsy Database 2.0 ( http://gydb.org ).

    Techniques Used: Sequencing, Produced

    PCR analysis to identify male and female seedlings of Cannabis sativa . In female plants, a band of approximately 540 bp in size was observed, while in male plants, a 390 bp size band was always observed and the 540 bp band was sometimes detected. (A,B) Strain “Moby Dyck” and “Blue Deity” showed a 5:7 and 9:5 ratio of male (M) and female (F) plants, respectively, from seeds derived from a male:female cross. (C) Strain “Healer” showed a 2:14 ratio of male:female plants. (D,E) All female plants derived from seeds resulting from hermaphroditic flowers of strains “Moby Dyck” (D) and “Space Queen” (E) . (F) PCR analysis of anther tissues (A) showing female composition compared to male (M) and female (F) plants. Water control with no DNA (C) and 1 kb DNA ladder (NEB Quick-Load ® ) (L).
    Figure Legend Snippet: PCR analysis to identify male and female seedlings of Cannabis sativa . In female plants, a band of approximately 540 bp in size was observed, while in male plants, a 390 bp size band was always observed and the 540 bp band was sometimes detected. (A,B) Strain “Moby Dyck” and “Blue Deity” showed a 5:7 and 9:5 ratio of male (M) and female (F) plants, respectively, from seeds derived from a male:female cross. (C) Strain “Healer” showed a 2:14 ratio of male:female plants. (D,E) All female plants derived from seeds resulting from hermaphroditic flowers of strains “Moby Dyck” (D) and “Space Queen” (E) . (F) PCR analysis of anther tissues (A) showing female composition compared to male (M) and female (F) plants. Water control with no DNA (C) and 1 kb DNA ladder (NEB Quick-Load ® ) (L).

    Techniques Used: Polymerase Chain Reaction, Derivative Assay

    Related Articles

    Amplification:

    Article Title: Characterisation of resistance mechanisms developed by basal cell carcinoma cells in response to repeated cycles of Photodynamic Therapy
    Article Snippet: .. The amplification products were purified with Monarch PCR & DNA Cleanup Kit (NEB) following the manufacturer’s recommendations. .. Fragments were sequenced in a capillary electrophoresis instrument AB3730XL (Applied Biosystems).

    Agarose Gel Electrophoresis:

    Article Title: Selection of an Efficient AAV Vector for Robust CNS Transgene Expression
    Article Snippet: .. The amplicons were then purified (Monarch PCR and DNA cleanup kit, New England Biolabs), digested by KpnI, AgeI, and BanII, and the Cap9 KpnI-AgeI fragments (144 bp) were agarose gel purified (Monarch DNA gel extraction kit, New England Biolabs) before ligation in the pUC57-Cap9-XbaI/AgeI/KpnI plasmid (opened with KpnI and AgeI and dephosphorylated with calf inositol phosphatase, New England Biolabs). .. The ligation products were transformed into electrocompetent DH5α bacteria (New England Biolabs), and the entire transformation was grown overnight in Lysogeny broth (LB)-ampicillin medium. pUC57-Cap9-XbaI/AgeI/KpnI plasmid was purified by Maxi Prep (QIAGEN).

    Ligation:

    Article Title: Selection of an Efficient AAV Vector for Robust CNS Transgene Expression
    Article Snippet: .. The amplicons were then purified (Monarch PCR and DNA cleanup kit, New England Biolabs), digested by KpnI, AgeI, and BanII, and the Cap9 KpnI-AgeI fragments (144 bp) were agarose gel purified (Monarch DNA gel extraction kit, New England Biolabs) before ligation in the pUC57-Cap9-XbaI/AgeI/KpnI plasmid (opened with KpnI and AgeI and dephosphorylated with calf inositol phosphatase, New England Biolabs). .. The ligation products were transformed into electrocompetent DH5α bacteria (New England Biolabs), and the entire transformation was grown overnight in Lysogeny broth (LB)-ampicillin medium. pUC57-Cap9-XbaI/AgeI/KpnI plasmid was purified by Maxi Prep (QIAGEN).

    Purification:

    Article Title: Characterisation of resistance mechanisms developed by basal cell carcinoma cells in response to repeated cycles of Photodynamic Therapy
    Article Snippet: .. The amplification products were purified with Monarch PCR & DNA Cleanup Kit (NEB) following the manufacturer’s recommendations. .. Fragments were sequenced in a capillary electrophoresis instrument AB3730XL (Applied Biosystems).

    Article Title: Selection of an Efficient AAV Vector for Robust CNS Transgene Expression
    Article Snippet: .. The amplicons were then purified (Monarch PCR and DNA cleanup kit, New England Biolabs), digested by KpnI, AgeI, and BanII, and the Cap9 KpnI-AgeI fragments (144 bp) were agarose gel purified (Monarch DNA gel extraction kit, New England Biolabs) before ligation in the pUC57-Cap9-XbaI/AgeI/KpnI plasmid (opened with KpnI and AgeI and dephosphorylated with calf inositol phosphatase, New England Biolabs). .. The ligation products were transformed into electrocompetent DH5α bacteria (New England Biolabs), and the entire transformation was grown overnight in Lysogeny broth (LB)-ampicillin medium. pUC57-Cap9-XbaI/AgeI/KpnI plasmid was purified by Maxi Prep (QIAGEN).

    Article Title: A fly model establishes distinct mechanisms for synthetic CRISPR/Cas9 sex distorters
    Article Snippet: .. The amplicons were purified with NEB Monarch PCR & DNA Cleanup Kit and quantified with Nanodrop. .. 200 ng were checked on a gel and 500 ng were sent to GENEWIZ to be sequenced with NGS-based amplicon sequencing.

    Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis
    Article Snippet: .. After 25 μl pilot PCRs, PCRs were scaled up (usually to 2 × 50 μl reactions) and gel purified using Monarch PCR and DNA cleanup kits (NEB). ..

    Article Title: DNA polymerase stalling at structured DNA constrains the expansion of short tandem repeats
    Article Snippet: .. The PCR products from all reactions were purified with the Monarch PCR & DNA Cleanup Kit (NEB) according to the manufacturer’s instructions. .. DNA sequencing libraries were then prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina according to the manufacturer’s instructions.

    Article Title: DNA polymerase stalling at structured DNA constrains the expansion of Short Tandem Repeats
    Article Snippet: .. The PCR products from all reactions were pooled and purified with the Monarch PCR & DNA Cleanup Kit (NEB) according to the manufacturer’s instructions. .. Ligation and transformation Purified DNA library (250 ng) was cut with the Hind III and Bam HI restriction enzymes (NEB) for 1h at 37 °C in a reaction mixture containing 1X of the NEB cut smart buffer.

    Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis
    Article Snippet: .. PCR products were purified using GeneJET PCR Purification Kits (Thermo Fisher Scientific, Waltham MA, USA), Nucleospin Gel and PCR Clean-up (Machery-Nagel GmbH, Düren, Germany) or Monarch PCR and DNA Cleanup kits (NEB). .. Gel purification was carried out using Monarch DNA Gel Extraction Kit (NEB).

    Polymerase Chain Reaction:

    Article Title: Characterisation of resistance mechanisms developed by basal cell carcinoma cells in response to repeated cycles of Photodynamic Therapy
    Article Snippet: .. The amplification products were purified with Monarch PCR & DNA Cleanup Kit (NEB) following the manufacturer’s recommendations. .. Fragments were sequenced in a capillary electrophoresis instrument AB3730XL (Applied Biosystems).

    Article Title: Selection of an Efficient AAV Vector for Robust CNS Transgene Expression
    Article Snippet: .. The amplicons were then purified (Monarch PCR and DNA cleanup kit, New England Biolabs), digested by KpnI, AgeI, and BanII, and the Cap9 KpnI-AgeI fragments (144 bp) were agarose gel purified (Monarch DNA gel extraction kit, New England Biolabs) before ligation in the pUC57-Cap9-XbaI/AgeI/KpnI plasmid (opened with KpnI and AgeI and dephosphorylated with calf inositol phosphatase, New England Biolabs). .. The ligation products were transformed into electrocompetent DH5α bacteria (New England Biolabs), and the entire transformation was grown overnight in Lysogeny broth (LB)-ampicillin medium. pUC57-Cap9-XbaI/AgeI/KpnI plasmid was purified by Maxi Prep (QIAGEN).

    Article Title: A fly model establishes distinct mechanisms for synthetic CRISPR/Cas9 sex distorters
    Article Snippet: .. The amplicons were purified with NEB Monarch PCR & DNA Cleanup Kit and quantified with Nanodrop. .. 200 ng were checked on a gel and 500 ng were sent to GENEWIZ to be sequenced with NGS-based amplicon sequencing.

    Article Title: Novel ssDNA Ligand Against Ovarian Cancer Biomarker CA125 With Promising Diagnostic Potential
    Article Snippet: .. Monarch PCR & DNA clean up kit (5 μg) and Monarch DNA gel extraction kit was purchased from New England Biolabs, India. .. Hot start Taq Polymerase was procured from Thermo Fisher Scientific, and all membranes were purchased from MDI, India.

    Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis
    Article Snippet: .. After 25 μl pilot PCRs, PCRs were scaled up (usually to 2 × 50 μl reactions) and gel purified using Monarch PCR and DNA cleanup kits (NEB). ..

    Article Title: DNA polymerase stalling at structured DNA constrains the expansion of short tandem repeats
    Article Snippet: .. The PCR products from all reactions were purified with the Monarch PCR & DNA Cleanup Kit (NEB) according to the manufacturer’s instructions. .. DNA sequencing libraries were then prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina according to the manufacturer’s instructions.

    Article Title: DNA polymerase stalling at structured DNA constrains the expansion of Short Tandem Repeats
    Article Snippet: .. The PCR products from all reactions were pooled and purified with the Monarch PCR & DNA Cleanup Kit (NEB) according to the manufacturer’s instructions. .. Ligation and transformation Purified DNA library (250 ng) was cut with the Hind III and Bam HI restriction enzymes (NEB) for 1h at 37 °C in a reaction mixture containing 1X of the NEB cut smart buffer.

    Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis
    Article Snippet: .. PCR products were purified using GeneJET PCR Purification Kits (Thermo Fisher Scientific, Waltham MA, USA), Nucleospin Gel and PCR Clean-up (Machery-Nagel GmbH, Düren, Germany) or Monarch PCR and DNA Cleanup kits (NEB). .. Gel purification was carried out using Monarch DNA Gel Extraction Kit (NEB).

    Gel Extraction:

    Article Title: Selection of an Efficient AAV Vector for Robust CNS Transgene Expression
    Article Snippet: .. The amplicons were then purified (Monarch PCR and DNA cleanup kit, New England Biolabs), digested by KpnI, AgeI, and BanII, and the Cap9 KpnI-AgeI fragments (144 bp) were agarose gel purified (Monarch DNA gel extraction kit, New England Biolabs) before ligation in the pUC57-Cap9-XbaI/AgeI/KpnI plasmid (opened with KpnI and AgeI and dephosphorylated with calf inositol phosphatase, New England Biolabs). .. The ligation products were transformed into electrocompetent DH5α bacteria (New England Biolabs), and the entire transformation was grown overnight in Lysogeny broth (LB)-ampicillin medium. pUC57-Cap9-XbaI/AgeI/KpnI plasmid was purified by Maxi Prep (QIAGEN).

    Article Title: Novel ssDNA Ligand Against Ovarian Cancer Biomarker CA125 With Promising Diagnostic Potential
    Article Snippet: .. Monarch PCR & DNA clean up kit (5 μg) and Monarch DNA gel extraction kit was purchased from New England Biolabs, India. .. Hot start Taq Polymerase was procured from Thermo Fisher Scientific, and all membranes were purchased from MDI, India.

    Plasmid Preparation:

    Article Title: Selection of an Efficient AAV Vector for Robust CNS Transgene Expression
    Article Snippet: .. The amplicons were then purified (Monarch PCR and DNA cleanup kit, New England Biolabs), digested by KpnI, AgeI, and BanII, and the Cap9 KpnI-AgeI fragments (144 bp) were agarose gel purified (Monarch DNA gel extraction kit, New England Biolabs) before ligation in the pUC57-Cap9-XbaI/AgeI/KpnI plasmid (opened with KpnI and AgeI and dephosphorylated with calf inositol phosphatase, New England Biolabs). .. The ligation products were transformed into electrocompetent DH5α bacteria (New England Biolabs), and the entire transformation was grown overnight in Lysogeny broth (LB)-ampicillin medium. pUC57-Cap9-XbaI/AgeI/KpnI plasmid was purified by Maxi Prep (QIAGEN).

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    New England Biolabs monarch pcr dna
    Serum stability profiling: (A) <t>PCR</t> amplified <t>DNA</t> and (B) its single-stranded form before amplification, after treatment with 50%v/v normal human female serum. Effect of different salt concentrations on aptamer-CA125 binding: (C) negative controls and (D) treated aptamer (lane 1: ladder, lane 2: 0.2 M NaHCO 3 with 0.5 M NaCl, lane 3: 100 mM NaCl and 5 mM MgCl 2 , lane 4: milli-Q water as positive control; (E) Binding—saturation curve for determination of K D . The original gel images have been provided in Figures S4, S5 .
    Monarch Pcr Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Serum stability profiling: (A) PCR amplified DNA and (B) its single-stranded form before amplification, after treatment with 50%v/v normal human female serum. Effect of different salt concentrations on aptamer-CA125 binding: (C) negative controls and (D) treated aptamer (lane 1: ladder, lane 2: 0.2 M NaHCO 3 with 0.5 M NaCl, lane 3: 100 mM NaCl and 5 mM MgCl 2 , lane 4: milli-Q water as positive control; (E) Binding—saturation curve for determination of K D . The original gel images have been provided in Figures S4, S5 .

    Journal: Frontiers in Chemistry

    Article Title: Novel ssDNA Ligand Against Ovarian Cancer Biomarker CA125 With Promising Diagnostic Potential

    doi: 10.3389/fchem.2020.00400

    Figure Lengend Snippet: Serum stability profiling: (A) PCR amplified DNA and (B) its single-stranded form before amplification, after treatment with 50%v/v normal human female serum. Effect of different salt concentrations on aptamer-CA125 binding: (C) negative controls and (D) treated aptamer (lane 1: ladder, lane 2: 0.2 M NaHCO 3 with 0.5 M NaCl, lane 3: 100 mM NaCl and 5 mM MgCl 2 , lane 4: milli-Q water as positive control; (E) Binding—saturation curve for determination of K D . The original gel images have been provided in Figures S4, S5 .

    Article Snippet: Monarch PCR & DNA clean up kit (5 μg) and Monarch DNA gel extraction kit was purchased from New England Biolabs, India.

    Techniques: Polymerase Chain Reaction, Amplification, Binding Assay, Positive Control

    (A) Assay to detect CRISPR/Cas9-mediated cleavage in vitro . A typical region of the Muc14a gene containing at least 2 binding sites for each of the gRNAs: Muc14a _3, Muc14a_4 , Muc14a_5 and Muc14a_6 (top). The PCR amplified DNA fragment was used as a digestion target for Cas9/gRNA cleavage reactions in vitro (bottom). Reactions were run on a gel to detect cleavage. A control without gRNA was included. (B) Analysis of combinations of gRNAs and Cas9 sources for X-shredding. Average male frequencies in the F1 progeny are shown for each parental genotype with a single copy of βtub85Dtub85D-cas9 transgene (1X), two copies of βtub85Dtub85D-cas9 transgene (2X) or one copy of nos-cas9 (grey bars). All lines were crossed to wild type w individuals. The reciprocal cross (female ctrl) or heterozygote βtub85Dtub85D-cas9/ + or nos-cas9/ + without gRNA (no gRNA) were used as control. The black arrow indicates gRNAs in the multiplex array and the red dotted line indicates an unbiased sex-ratio. Crosses were set as pools of males and females or as multiple male single crosses in which case error bars indicate the mean ± SD for a minimum of ten independent single crosses. For all crosses n indicates the total number of individuals (males + females) in the F1 progeny counted. (C) Developmental survival analysis of the F1 progeny of Muc14a_6/βtub85Dtub85D-cas9 males crossed to w females compared to w and βtub85Dtub85D-cas9/ + control males crossed to w females. n indicates the number of individuals recorded at every developmental stage (males + females) in the F1 progeny. Bars indicate means ± SD for at least ten independent single crosses. Statistical significance was calculated with a t test assuming unequal variance. ** p

    Journal: bioRxiv

    Article Title: A fly model establishes distinct mechanisms for synthetic CRISPR/Cas9 sex distorters

    doi: 10.1101/834630

    Figure Lengend Snippet: (A) Assay to detect CRISPR/Cas9-mediated cleavage in vitro . A typical region of the Muc14a gene containing at least 2 binding sites for each of the gRNAs: Muc14a _3, Muc14a_4 , Muc14a_5 and Muc14a_6 (top). The PCR amplified DNA fragment was used as a digestion target for Cas9/gRNA cleavage reactions in vitro (bottom). Reactions were run on a gel to detect cleavage. A control without gRNA was included. (B) Analysis of combinations of gRNAs and Cas9 sources for X-shredding. Average male frequencies in the F1 progeny are shown for each parental genotype with a single copy of βtub85Dtub85D-cas9 transgene (1X), two copies of βtub85Dtub85D-cas9 transgene (2X) or one copy of nos-cas9 (grey bars). All lines were crossed to wild type w individuals. The reciprocal cross (female ctrl) or heterozygote βtub85Dtub85D-cas9/ + or nos-cas9/ + without gRNA (no gRNA) were used as control. The black arrow indicates gRNAs in the multiplex array and the red dotted line indicates an unbiased sex-ratio. Crosses were set as pools of males and females or as multiple male single crosses in which case error bars indicate the mean ± SD for a minimum of ten independent single crosses. For all crosses n indicates the total number of individuals (males + females) in the F1 progeny counted. (C) Developmental survival analysis of the F1 progeny of Muc14a_6/βtub85Dtub85D-cas9 males crossed to w females compared to w and βtub85Dtub85D-cas9/ + control males crossed to w females. n indicates the number of individuals recorded at every developmental stage (males + females) in the F1 progeny. Bars indicate means ± SD for at least ten independent single crosses. Statistical significance was calculated with a t test assuming unequal variance. ** p

    Article Snippet: The amplicons were purified with NEB Monarch PCR & DNA Cleanup Kit and quantified with Nanodrop.

    Techniques: CRISPR, In Vitro, Binding Assay, Polymerase Chain Reaction, Amplification, Multiplex Assay

    Principles of Darwin Assembly. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the cut strand degraded by exonuclease III (1). Boundary and inner (mutagenic) oligonucleotides are annealed to the ssDNA plasmid (2). Key features of the oligonucleotides are highlighted: 5′-boundary oligonucleotide is 5′-biotinylated; non-complementary overhangs are shown in blue with Type IIs endonuclease recognition sites shown in white; mutations are shown as red X in the inner oligonucleotides; 3′-boundary oligonucleotide is protected at its 3′-end. After annealing, primers are extended and ligated in an isothermal assembly reaction (3). The assembled strand can be isolated by paramagnetic streptavidin-coated beads (4) and purified by alkali washing prior to PCR using outnested priming sites (5) and cloning (6) using the type IIS restriction sites (white dots). The purification step (4) is not always necessary but we found it improved PCR performance, especially for long assembly reactions ( > 1 kb).

    Journal: Nucleic Acids Research

    Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

    doi: 10.1093/nar/gky067

    Figure Lengend Snippet: Principles of Darwin Assembly. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the cut strand degraded by exonuclease III (1). Boundary and inner (mutagenic) oligonucleotides are annealed to the ssDNA plasmid (2). Key features of the oligonucleotides are highlighted: 5′-boundary oligonucleotide is 5′-biotinylated; non-complementary overhangs are shown in blue with Type IIs endonuclease recognition sites shown in white; mutations are shown as red X in the inner oligonucleotides; 3′-boundary oligonucleotide is protected at its 3′-end. After annealing, primers are extended and ligated in an isothermal assembly reaction (3). The assembled strand can be isolated by paramagnetic streptavidin-coated beads (4) and purified by alkali washing prior to PCR using outnested priming sites (5) and cloning (6) using the type IIS restriction sites (white dots). The purification step (4) is not always necessary but we found it improved PCR performance, especially for long assembly reactions ( > 1 kb).

    Article Snippet: PCR products were purified using GeneJET PCR Purification Kits (Thermo Fisher Scientific, Waltham MA, USA), Nucleospin Gel and PCR Clean-up (Machery-Nagel GmbH, Düren, Germany) or Monarch PCR and DNA Cleanup kits (NEB).

    Techniques: Plasmid Preparation, Isolation, Purification, Polymerase Chain Reaction, Clone Assay

    Darwin Assembly using a θ oligonucleotide. Here, a single θ oligonucleotide is used in place of the two boundary oligonucleotides allowing enzymatic cleanup after the assembly reaction. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the nicked strand degraded by exonuclease III (1). Inner oligonucleotides and a single θ oligonucleotide are annealed to the ssDNA plasmid (2). The θ oligonucleotide encodes both assembly priming and termination sequences linked by a flexible linker such that successful assembly of the mutated strand results in a closed circle (3). The template plasmid can now be linearized (e.g. at the yellow dot, by adding a targeting oligonucleotide and appropriate restriction endonuclease) and both exonuclease I and exonuclease III added to degrade any non-circular DNA (4). The mutated gene can now be amplified from the closed circle by PCR (5) and cloned into a fresh vector (6) using the type IIS restriction sites (white dots).

    Journal: Nucleic Acids Research

    Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

    doi: 10.1093/nar/gky067

    Figure Lengend Snippet: Darwin Assembly using a θ oligonucleotide. Here, a single θ oligonucleotide is used in place of the two boundary oligonucleotides allowing enzymatic cleanup after the assembly reaction. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the nicked strand degraded by exonuclease III (1). Inner oligonucleotides and a single θ oligonucleotide are annealed to the ssDNA plasmid (2). The θ oligonucleotide encodes both assembly priming and termination sequences linked by a flexible linker such that successful assembly of the mutated strand results in a closed circle (3). The template plasmid can now be linearized (e.g. at the yellow dot, by adding a targeting oligonucleotide and appropriate restriction endonuclease) and both exonuclease I and exonuclease III added to degrade any non-circular DNA (4). The mutated gene can now be amplified from the closed circle by PCR (5) and cloned into a fresh vector (6) using the type IIS restriction sites (white dots).

    Article Snippet: PCR products were purified using GeneJET PCR Purification Kits (Thermo Fisher Scientific, Waltham MA, USA), Nucleospin Gel and PCR Clean-up (Machery-Nagel GmbH, Düren, Germany) or Monarch PCR and DNA Cleanup kits (NEB).

    Techniques: Plasmid Preparation, Amplification, Polymerase Chain Reaction, Clone Assay

    Darwin assembled TgoT DNA polymerase library. ( A ) Five separate sequencing reactions (range and reads shown in blue) were required to sample the diversity introduced across the eight target residues (shown in red along the TgoT gene). Mutations included focused degeneracies (e.g. YWC used against Y384) or ‘small intelligent’ (S-int) diversity (NDT, VMA, ATG and TGG oligonucleotides mixed in a 12:6:1:1 ratio). Resulting incorporation is shown in box plots with outliers explicitly labelled. Wild-type contamination was determined from positions where diversity excluded those sequences (N.A.: not applicable). As with the T7 RNA polymerase library, incorporation trends and biases were analysed to identify any biases in assembly. Ranked incorporation frequencies are shown for the residues targeted with ‘small intelligent’ diversity, and the top three highest (greens) and lowest (oranges) ranked codons (based on a straight sum of ranks) are highlighted ( B ). Outnest PCR of the TgoT DNA polymerase library (expected product of 2501 bp) showing that the final PCR can be carried out with either A- (MyTaq) or B-family (Q5) polymerases ( C ). MW: 1 kb ladder (NEB). NT: no template PCR control.

    Journal: Nucleic Acids Research

    Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

    doi: 10.1093/nar/gky067

    Figure Lengend Snippet: Darwin assembled TgoT DNA polymerase library. ( A ) Five separate sequencing reactions (range and reads shown in blue) were required to sample the diversity introduced across the eight target residues (shown in red along the TgoT gene). Mutations included focused degeneracies (e.g. YWC used against Y384) or ‘small intelligent’ (S-int) diversity (NDT, VMA, ATG and TGG oligonucleotides mixed in a 12:6:1:1 ratio). Resulting incorporation is shown in box plots with outliers explicitly labelled. Wild-type contamination was determined from positions where diversity excluded those sequences (N.A.: not applicable). As with the T7 RNA polymerase library, incorporation trends and biases were analysed to identify any biases in assembly. Ranked incorporation frequencies are shown for the residues targeted with ‘small intelligent’ diversity, and the top three highest (greens) and lowest (oranges) ranked codons (based on a straight sum of ranks) are highlighted ( B ). Outnest PCR of the TgoT DNA polymerase library (expected product of 2501 bp) showing that the final PCR can be carried out with either A- (MyTaq) or B-family (Q5) polymerases ( C ). MW: 1 kb ladder (NEB). NT: no template PCR control.

    Article Snippet: PCR products were purified using GeneJET PCR Purification Kits (Thermo Fisher Scientific, Waltham MA, USA), Nucleospin Gel and PCR Clean-up (Machery-Nagel GmbH, Düren, Germany) or Monarch PCR and DNA Cleanup kits (NEB).

    Techniques: Sequencing, Polymerase Chain Reaction