monarch pcr dna cleanup kit  (New England Biolabs)


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    Name:
    Monarch PCR DNA Cleanup Kit
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    Catalog Number:
    T1030
    Price:
    475
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    Applications:
    DNA Manipulation
    Size:
    250 preps
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    Structured Review

    New England Biolabs monarch pcr dna cleanup kit
    Monarch PCR DNA Cleanup Kit

    https://www.bioz.com/result/monarch pcr dna cleanup kit/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    monarch pcr dna cleanup kit - by Bioz Stars, 2021-10
    99/100 stars

    Images

    1) Product Images from "The FKH domain in FOXP3 mRNA frequently contains mutations in hepatocellular carcinoma that influence the subcellular localization and functions of FOXP3"

    Article Title: The FKH domain in FOXP3 mRNA frequently contains mutations in hepatocellular carcinoma that influence the subcellular localization and functions of FOXP3

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA120.012518

    Mutations were detected in FKH domains of FOXP3 transcripts in HCC. A , representative sequencing chromatograms of point mutations. B , representative sequencing chromatograms of the complicated mutation status. Upper left panel , FKH sequences in mRNA. Upper right panel , sequences of the corresponding regions in genomic DNA. Lower panel , sequences of individual TA clones which were generated to examine individual sequences in multiple-mutation bearing PCR products. C , representative immunohistochemical results of FOXP3-positive lymphocyte distribution in tumors and corresponding nontumorous tissues. Black arrow , FOXP3-positive lymphocytes.
    Figure Legend Snippet: Mutations were detected in FKH domains of FOXP3 transcripts in HCC. A , representative sequencing chromatograms of point mutations. B , representative sequencing chromatograms of the complicated mutation status. Upper left panel , FKH sequences in mRNA. Upper right panel , sequences of the corresponding regions in genomic DNA. Lower panel , sequences of individual TA clones which were generated to examine individual sequences in multiple-mutation bearing PCR products. C , representative immunohistochemical results of FOXP3-positive lymphocyte distribution in tumors and corresponding nontumorous tissues. Black arrow , FOXP3-positive lymphocytes.

    Techniques Used: Sequencing, Mutagenesis, Clone Assay, Generated, Polymerase Chain Reaction, Immunohistochemistry

    2) Product Images from "Duplex Proximity Sequencing (Pro-Seq): A method to improve DNA sequencing accuracy without the cost of molecular barcoding redundancy"

    Article Title: Duplex Proximity Sequencing (Pro-Seq): A method to improve DNA sequencing accuracy without the cost of molecular barcoding redundancy

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0204265

    Overview of the targeted Pro-Seq workflow (described in detail in the Materials and methods ). In brief, double stranded DNA is loaded directly into droplets such that on average zero or one template molecule is incorporated in each droplet. Off-target DNA (not shown in figure) is also loaded into droplets, but does not amplify. Within each droplet are multiplexed gene-specific primers, and the Pro-Seq universal 5’ PEG-linked primers. The droplets are PCR cycled such that all copies of the starting template are linked to the universal linked primers (shown in detail in S2 Fig ). The emulsions are then broken, and the un-linked strands are digested and cleaned up. After quantification, the library is ready for sequencing.
    Figure Legend Snippet: Overview of the targeted Pro-Seq workflow (described in detail in the Materials and methods ). In brief, double stranded DNA is loaded directly into droplets such that on average zero or one template molecule is incorporated in each droplet. Off-target DNA (not shown in figure) is also loaded into droplets, but does not amplify. Within each droplet are multiplexed gene-specific primers, and the Pro-Seq universal 5’ PEG-linked primers. The droplets are PCR cycled such that all copies of the starting template are linked to the universal linked primers (shown in detail in S2 Fig ). The emulsions are then broken, and the un-linked strands are digested and cleaned up. After quantification, the library is ready for sequencing.

    Techniques Used: Polymerase Chain Reaction, Sequencing

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

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

    Journal: Nature Communications

    doi: 10.1038/s41467-021-24295-2

    Screen for 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 Motif logos generated from aligned hexamers that were enriched by Bicc1-FLAG. d 4-mer, 5-mer, and 6-mer sequences ranked by their relative frequencies in Bicc1-FLAG versus control RBNS data. e Maps of GAC-containing motifs in the 3′-UTR of Dand5 mRNAs for the indicated species. f Schematic representation of metagene analysis for the 200-nucleotide proximal region of the 3′-UTR of mouse mRNAs. A total of 31,165 regions extracted from mouse genes (mm10) was searched with the indicated target motifs. g Histogram of motif frequency revealed by metagene analysis. The vertical lines indicate the averaged frequency of each target motif (black) and the frequency of each target motif in the 200-nucleotide proximal region of the 3′-UTR of Dand5 mRNA (blue), respectively. h Multiple sequence alignment of a conserved segment within the proximal 200 nucleotides of mammalian, amphibian, and fish Dand5 3′ -UTRs . Colors highlight GAC motifs.
    Figure Legend Snippet: Screen for 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 Motif logos generated from aligned hexamers that were enriched by Bicc1-FLAG. d 4-mer, 5-mer, and 6-mer sequences ranked by their relative frequencies in Bicc1-FLAG versus control RBNS data. e Maps of GAC-containing motifs in the 3′-UTR of Dand5 mRNAs for the indicated species. f Schematic representation of metagene analysis for the 200-nucleotide proximal region of the 3′-UTR of mouse mRNAs. A total of 31,165 regions extracted from mouse genes (mm10) was searched with the indicated target motifs. g Histogram of motif frequency revealed by metagene analysis. The vertical lines indicate the averaged frequency of each target motif (black) and the frequency of each target motif in the 200-nucleotide proximal region of the 3′-UTR of Dand5 mRNA (blue), respectively. h Multiple sequence alignment of a conserved segment within the proximal 200 nucleotides of mammalian, amphibian, and fish Dand5 3′ -UTRs . Colors highlight GAC motifs.

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

    4) Product Images from "Using weapons instead of perfume – chemical association strategies of the myrmecophilous bug Scolopostethus pacificus (Rhyparochromidae)"

    Article Title: Using weapons instead of perfume – chemical association strategies of the myrmecophilous bug Scolopostethus pacificus (Rhyparochromidae)

    Journal: bioRxiv

    doi: 10.1101/2020.12.08.412577

    Molecular gut content analysis of Scolopostethus pacificus . Standard ITS2 (CAS5ps+CAS28s) and ant-specific Loc1 and Loc2 primers were used for PCR amplification using S. pacificus body-DNA, gut-DNA and Liometopum occidentale -DNA. While ITS2 amplified in all cases, no ITS2 fragment was amplified with ant-specific ITS (Loc 1/2) was amplified from dissected bugs’ guts.
    Figure Legend Snippet: Molecular gut content analysis of Scolopostethus pacificus . Standard ITS2 (CAS5ps+CAS28s) and ant-specific Loc1 and Loc2 primers were used for PCR amplification using S. pacificus body-DNA, gut-DNA and Liometopum occidentale -DNA. While ITS2 amplified in all cases, no ITS2 fragment was amplified with ant-specific ITS (Loc 1/2) was amplified from dissected bugs’ guts.

    Techniques Used: Polymerase Chain Reaction, Amplification

    5) 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

    6) 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

    7) Product Images from "Bisulfite-free epigenomics and genomics of single cells through methylation-sensitive restriction"

    Article Title: Bisulfite-free epigenomics and genomics of single cells through methylation-sensitive restriction

    Journal: Communications Biology

    doi: 10.1038/s42003-021-01661-w

    epi-gSCAR workflow schematics and methylation readout for the DLX4 locus. a epi-gSCAR workflow: single-cell isolation is followed by lysis and chromatin digestion to render DNA accessible for methylation-sensitive restriction enzyme (MSRE) digestion with HhaI (i). Cleavage of methylated HhaI sites (light blue) is blocked, while unmethylated sites (dark blue) are cleaved; the resulting DNA ends are tagged with poly(d)A tails (red) (ii). Poly(d)A tails are primed by anchored (GAT-oligo(dT)12-CG, blue–green–gray, assay variant A) or non-anchored adapters (GAT-oligo(dT)12, green–gray, assay variant B). Anchored adapters were used to limit the length of poly(d)A tails in the library (Supplementary Fig. 8 ). This is followed by gap filling and ligation, which results in tagged restriction enzyme scars (iii). Random priming by 7N-GAT adapters (orange–gray) facilitates quasilinear amplification of the genome (iv). PCR generates amplicons carrying genetic and epigenetic information (v). b HhaI sites in CGIs and around TSSs across 100 bp windows and 3 kb upstream and downstream. c Methylation analysis of the DLX4 locus by step-out PCR followed by Sanger sequencing. DLX4 locus with CGI1 (green) and CGI2 (red), CpGs (red) and HhaI sites (blue), primer map for analysis of HhaI sites 1–10 (CGI1) and 3–21 (CGI2), and corresponding sequencing reads. Magnification of reads obtained from single cell K_05 (selected for analysis as it demonstrated satisfactory results in initial suppression PCR experiments) corresponding to HhaI sites 4–6 in CGI2 showing intact and tagged-scar HhaI sites: intact HhaI sites are called as having been methylated and poly(d)A-tailed HhaI scar sites unmethylated; presence of both suggests heterozygous methylation. d DNA methylation in single cells K_01–K_07 at individual HhaI sites for CGI1 (green) and CGI2 (red) of DLX4 assessed by PCR and/or NGS (Supplementary Fig. 3 ), and comparison with Kasumi-1 cell-bulk whole-genome bisulfite sequencing (WGBS) data. Using step-out PCR on single cell K_05, CGI1 was unmethylated at all analyzed HhaI sites (6/6). CGI2 featured high level of heterozygous methylation (14/19 methylated; 5/19 heterozygous methylation). e Mean methylation levels of CGI1 (green) and CGI2 (red) for single cells K_01–K_07 (NGS and PCR), Kasumi-1 WGBS and Illumina 450 K array.
    Figure Legend Snippet: epi-gSCAR workflow schematics and methylation readout for the DLX4 locus. a epi-gSCAR workflow: single-cell isolation is followed by lysis and chromatin digestion to render DNA accessible for methylation-sensitive restriction enzyme (MSRE) digestion with HhaI (i). Cleavage of methylated HhaI sites (light blue) is blocked, while unmethylated sites (dark blue) are cleaved; the resulting DNA ends are tagged with poly(d)A tails (red) (ii). Poly(d)A tails are primed by anchored (GAT-oligo(dT)12-CG, blue–green–gray, assay variant A) or non-anchored adapters (GAT-oligo(dT)12, green–gray, assay variant B). Anchored adapters were used to limit the length of poly(d)A tails in the library (Supplementary Fig. 8 ). This is followed by gap filling and ligation, which results in tagged restriction enzyme scars (iii). Random priming by 7N-GAT adapters (orange–gray) facilitates quasilinear amplification of the genome (iv). PCR generates amplicons carrying genetic and epigenetic information (v). b HhaI sites in CGIs and around TSSs across 100 bp windows and 3 kb upstream and downstream. c Methylation analysis of the DLX4 locus by step-out PCR followed by Sanger sequencing. DLX4 locus with CGI1 (green) and CGI2 (red), CpGs (red) and HhaI sites (blue), primer map for analysis of HhaI sites 1–10 (CGI1) and 3–21 (CGI2), and corresponding sequencing reads. Magnification of reads obtained from single cell K_05 (selected for analysis as it demonstrated satisfactory results in initial suppression PCR experiments) corresponding to HhaI sites 4–6 in CGI2 showing intact and tagged-scar HhaI sites: intact HhaI sites are called as having been methylated and poly(d)A-tailed HhaI scar sites unmethylated; presence of both suggests heterozygous methylation. d DNA methylation in single cells K_01–K_07 at individual HhaI sites for CGI1 (green) and CGI2 (red) of DLX4 assessed by PCR and/or NGS (Supplementary Fig. 3 ), and comparison with Kasumi-1 cell-bulk whole-genome bisulfite sequencing (WGBS) data. Using step-out PCR on single cell K_05, CGI1 was unmethylated at all analyzed HhaI sites (6/6). CGI2 featured high level of heterozygous methylation (14/19 methylated; 5/19 heterozygous methylation). e Mean methylation levels of CGI1 (green) and CGI2 (red) for single cells K_01–K_07 (NGS and PCR), Kasumi-1 WGBS and Illumina 450 K array.

    Techniques Used: Methylation, Single-cell Isolation, Lysis, Variant Assay, Ligation, Amplification, Polymerase Chain Reaction, Sequencing, DNA Methylation Assay, Next-Generation Sequencing, Methylation Sequencing

    8) Product Images from "Efficient coralline algal psbA mini barcoding and High Resolution Melt (HRM) analysis using a simple custom DNA preparation"

    Article Title: Efficient coralline algal psbA mini barcoding and High Resolution Melt (HRM) analysis using a simple custom DNA preparation

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-36998-6

    DNA preparation (3 methods) and PCR (4 protocols) comparisons. 1 Kb DNA ladder (L). Grinded sample with QuickExtract followed with Monarch purification, eluate diluted 10 −1 in Di H 2 O (A1, A2, A3 A4). Whole sample with QuickExtract, eluate diluted 10 −2 in Di H 2 O (B1, B2, B3 B4). Whole sample with QuickExtract followed with Monarch purification, eluate diluted 10 −1 in Di H 2 O (C1, C2, C3 C4). Primers GazF1 GazR1 producing a 610 bp amplicon (A1, B1 C1). Primers psbA21-350F psbA22-350R producing a 350 bp amplicon (A2, B2 C2). Primers psbA-F1 psbA-R1 producing a 957 bp amplicon (A3, B3 C3). Primers psbA-F1 psbA-R600 producing a 600 bp amplicon (A4, B4 C4). The full-length gel is presented in Supplementary Figure S3 .
    Figure Legend Snippet: DNA preparation (3 methods) and PCR (4 protocols) comparisons. 1 Kb DNA ladder (L). Grinded sample with QuickExtract followed with Monarch purification, eluate diluted 10 −1 in Di H 2 O (A1, A2, A3 A4). Whole sample with QuickExtract, eluate diluted 10 −2 in Di H 2 O (B1, B2, B3 B4). Whole sample with QuickExtract followed with Monarch purification, eluate diluted 10 −1 in Di H 2 O (C1, C2, C3 C4). Primers GazF1 GazR1 producing a 610 bp amplicon (A1, B1 C1). Primers psbA21-350F psbA22-350R producing a 350 bp amplicon (A2, B2 C2). Primers psbA-F1 psbA-R1 producing a 957 bp amplicon (A3, B3 C3). Primers psbA-F1 psbA-R600 producing a 600 bp amplicon (A4, B4 C4). The full-length gel is presented in Supplementary Figure S3 .

    Techniques Used: Polymerase Chain Reaction, Purification, Amplification

    9) 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

    10) 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

    11) Product Images from "CRISPR-Cas12a-assisted PCR tagging of mammalian genes"

    Article Title: CRISPR-Cas12a-assisted PCR tagging of mammalian genes

    Journal: bioRxiv

    doi: 10.1101/473876

    PCR Strategy PCR is performed with M1 and M2 tagging oligos and a template cassette that contains the tag. The M1 and M2 tagging oligos provide the homology arms (HA, ∼55 to 90 nt in length) for targeted integration. The M2 tagging oligo additionally provides the direct repeat and a protospacer sequence (orange) for a Cas12a endonuclease. The template cassette contains the desired tag and additional features, such as a selection marker. It also contains the U6 Pol III promoter for driving crRNA expression. PCR yields a linear DNA fragment (PCR cassette) that contains homology arms to the target locus and a functional crRNA gene to cleave the locus.
    Figure Legend Snippet: PCR Strategy PCR is performed with M1 and M2 tagging oligos and a template cassette that contains the tag. The M1 and M2 tagging oligos provide the homology arms (HA, ∼55 to 90 nt in length) for targeted integration. The M2 tagging oligo additionally provides the direct repeat and a protospacer sequence (orange) for a Cas12a endonuclease. The template cassette contains the desired tag and additional features, such as a selection marker. It also contains the U6 Pol III promoter for driving crRNA expression. PCR yields a linear DNA fragment (PCR cassette) that contains homology arms to the target locus and a functional crRNA gene to cleave the locus.

    Techniques Used: Polymerase Chain Reaction, Sequencing, Selection, Marker, Expressing, Functional Assay

    Tagging efficiency as a function of different parameters. ( a ) Length of homology arms. M1 and M2 tagging oligos containing the indicated sequence lengths of homology arm (5’-HA and 3’-HA, respectively) to the destination locus were used for PCR tagging of the HNRNPA1 locus in HEK293T cells. Tagging efficiency was estimated 3 days after transfection as described before. Data from three replicates is shown. Error bars indicate SD. ( b ) PCR cassettes containing various types of ends to direct the choice of DNA repair pathway: Homology arms (90-bp and 55-bp homology, for HR; A ), blunt ended arms without homology to the target locus (blunt; B ), Hga I cut ( D ) and uncut ends ( C ). Cutting with the type IIS restriction enzyme Hga I results in 5-nt 3’-overhangs that are complementary to the overhangs generated by the crRNA directed Cas12a-cleavage of the destination locus. Tagging efficiency was estimated three days later as described in (a) using HEK293T cells. Data from three replicates is shown. Error bars indicate SD. ( c ) Use of modified and/or purified oligos. M1/M2 tagging oligos with the indicated number of phosphorothioate bonds and/or biotin as indicated were used for generation of PCR cassettes. All oligos were ‘cartridge’ purified except for the ones denoted with ‘PAGE’, which were size selected using polyacrylamide gel electrophoresis. Tagging efficiency was estimated three days after transfection as described before using HEK293T cells. Data from three replicates is shown. Error bars indicate SD.
    Figure Legend Snippet: Tagging efficiency as a function of different parameters. ( a ) Length of homology arms. M1 and M2 tagging oligos containing the indicated sequence lengths of homology arm (5’-HA and 3’-HA, respectively) to the destination locus were used for PCR tagging of the HNRNPA1 locus in HEK293T cells. Tagging efficiency was estimated 3 days after transfection as described before. Data from three replicates is shown. Error bars indicate SD. ( b ) PCR cassettes containing various types of ends to direct the choice of DNA repair pathway: Homology arms (90-bp and 55-bp homology, for HR; A ), blunt ended arms without homology to the target locus (blunt; B ), Hga I cut ( D ) and uncut ends ( C ). Cutting with the type IIS restriction enzyme Hga I results in 5-nt 3’-overhangs that are complementary to the overhangs generated by the crRNA directed Cas12a-cleavage of the destination locus. Tagging efficiency was estimated three days later as described in (a) using HEK293T cells. Data from three replicates is shown. Error bars indicate SD. ( c ) Use of modified and/or purified oligos. M1/M2 tagging oligos with the indicated number of phosphorothioate bonds and/or biotin as indicated were used for generation of PCR cassettes. All oligos were ‘cartridge’ purified except for the ones denoted with ‘PAGE’, which were size selected using polyacrylamide gel electrophoresis. Tagging efficiency was estimated three days after transfection as described before using HEK293T cells. Data from three replicates is shown. Error bars indicate SD.

    Techniques Used: Sequencing, Polymerase Chain Reaction, Transfection, Generated, Modification, Purification, Polyacrylamide Gel Electrophoresis

    Endogenous C-terminal gene tagging in mammalian cells using PCR tagging. ( a ) For tag insertion before the STOP codon of an ORF, two ‘Gene specific tagging oligos’ (termed M1 and M2) are designed using an online tool ( www.pcr-tagging.com ). A ‘Tagging PCR’ with a generic ‘Template plasmid’ generates the gene-specific ‘PCR cassette’. The ‘Template plasmid’ provides the tag (e.g. a fluorescent protein), a possible selection marker and a Pol III promoter. For gene tagging the ‘PCR cassette’ is transfected into the target cell together with a helper plasmid containing a Cas12a endonuclease gene. This leads to insertion of the PCR cassette into the chromosome, which yields a fusion of the tag (e.g. GFP) with the target gene. ( b ) Tagging Principle: The PCR cassette contains a crRNA sequence that is expressed inside the cell via an U6 promoter (Pol III promoter). The crRNA directs Cas12a (which is expressed from the helper plasmid) to the target locus close to the insertion site. Stimulated by the DSB the linear PCR cassette is then inserted into the genome. The homology arm of the M1 tagging oligo thereby directs in frame fusion of the tag with the target ORF, leading to the expression of a tagged protein from the target locus. Integration leads to destruction of the crRNA target site, thus preventing re-cleavage of the modified locus. ( c ) Efficiency of C-terminal mNeonGreen-tagging for 16 organelle specific genes. For each gene, specific M1/M2 tagging oligos were used to amplify an mNeonGreen containing template plasmid. The resulting PCR cassettes were transfected in HEK293T cells. HOECHST staining of live cells and analysis by fluorescence microscopy was performed three days after transfection. Fractions of cells exhibiting the expected localization or diffuse cytoplasmic green fluorescence are shown. For information on selected genes, see Table S1. Data from one representative experiment is shown. ( d ) Representative images from HEK293T cells 3 days after transfection. mNeonGreen fluorescence and HOECHST staining (DNA) is shown. In addition to the expected localization, cells showing diffuse cytoplasmic fluorescence (arrows) are detected. ( e ) Tagging is specific for the crRNA and guided by the homology arms (HAs). Efficiency of control transfections (see Fig. S2 for representative examples). * in this transfection indicates that a matching combination of crRNA and HAs was used, but the crRNA was expressed from a different PCR fragment. ** indicates that in this case a PCR cassette was used where the crRNA (for CANX) lead to cleavage of a different gene than the one specified by the HAs (HNRNPA1). A small fraction of cells (
    Figure Legend Snippet: Endogenous C-terminal gene tagging in mammalian cells using PCR tagging. ( a ) For tag insertion before the STOP codon of an ORF, two ‘Gene specific tagging oligos’ (termed M1 and M2) are designed using an online tool ( www.pcr-tagging.com ). A ‘Tagging PCR’ with a generic ‘Template plasmid’ generates the gene-specific ‘PCR cassette’. The ‘Template plasmid’ provides the tag (e.g. a fluorescent protein), a possible selection marker and a Pol III promoter. For gene tagging the ‘PCR cassette’ is transfected into the target cell together with a helper plasmid containing a Cas12a endonuclease gene. This leads to insertion of the PCR cassette into the chromosome, which yields a fusion of the tag (e.g. GFP) with the target gene. ( b ) Tagging Principle: The PCR cassette contains a crRNA sequence that is expressed inside the cell via an U6 promoter (Pol III promoter). The crRNA directs Cas12a (which is expressed from the helper plasmid) to the target locus close to the insertion site. Stimulated by the DSB the linear PCR cassette is then inserted into the genome. The homology arm of the M1 tagging oligo thereby directs in frame fusion of the tag with the target ORF, leading to the expression of a tagged protein from the target locus. Integration leads to destruction of the crRNA target site, thus preventing re-cleavage of the modified locus. ( c ) Efficiency of C-terminal mNeonGreen-tagging for 16 organelle specific genes. For each gene, specific M1/M2 tagging oligos were used to amplify an mNeonGreen containing template plasmid. The resulting PCR cassettes were transfected in HEK293T cells. HOECHST staining of live cells and analysis by fluorescence microscopy was performed three days after transfection. Fractions of cells exhibiting the expected localization or diffuse cytoplasmic green fluorescence are shown. For information on selected genes, see Table S1. Data from one representative experiment is shown. ( d ) Representative images from HEK293T cells 3 days after transfection. mNeonGreen fluorescence and HOECHST staining (DNA) is shown. In addition to the expected localization, cells showing diffuse cytoplasmic fluorescence (arrows) are detected. ( e ) Tagging is specific for the crRNA and guided by the homology arms (HAs). Efficiency of control transfections (see Fig. S2 for representative examples). * in this transfection indicates that a matching combination of crRNA and HAs was used, but the crRNA was expressed from a different PCR fragment. ** indicates that in this case a PCR cassette was used where the crRNA (for CANX) lead to cleavage of a different gene than the one specified by the HAs (HNRNPA1). A small fraction of cells (

    Techniques Used: Polymerase Chain Reaction, Plasmid Preparation, Selection, Marker, Transfection, Sequencing, Expressing, Modification, Staining, Fluorescence, Microscopy

    Exploring transfection parameters ( a ) Impact of transfected amounts of DNA on tagging efficiency using HEK293T cells. Transfected amounts of PCR cassette and Cas12a plasmid as indicated. Always 1 µg of DNA was transfected using lipofectamine. pUC18 was used as neutral DNA. Tagging efficiency was determined three days later by HOECHST staining and live cell imaging. Data from one representative experiment is shown. ( b ) HEK293T cells were transfected for 4 hours or overnight using Lipofectamine 2000 or transfected using electroporation, as indicated. Tagging efficiency was determined three days later as described in (a). Data from one representative experiment is shown. ( c ) HEK293T cells were transfected in duplicates by electroporation with Cas12a protein, Cas12a-encoding mRNA or Cas12-encoding plasmid. For protein-based expression 100 ng of PCR cassette while for mRNA and plasmid 1.5 µg PCR cassette were electroporated. Error bars indicate range between the technical duplicates.
    Figure Legend Snippet: Exploring transfection parameters ( a ) Impact of transfected amounts of DNA on tagging efficiency using HEK293T cells. Transfected amounts of PCR cassette and Cas12a plasmid as indicated. Always 1 µg of DNA was transfected using lipofectamine. pUC18 was used as neutral DNA. Tagging efficiency was determined three days later by HOECHST staining and live cell imaging. Data from one representative experiment is shown. ( b ) HEK293T cells were transfected for 4 hours or overnight using Lipofectamine 2000 or transfected using electroporation, as indicated. Tagging efficiency was determined three days later as described in (a). Data from one representative experiment is shown. ( c ) HEK293T cells were transfected in duplicates by electroporation with Cas12a protein, Cas12a-encoding mRNA or Cas12-encoding plasmid. For protein-based expression 100 ng of PCR cassette while for mRNA and plasmid 1.5 µg PCR cassette were electroporated. Error bars indicate range between the technical duplicates.

    Techniques Used: Transfection, Polymerase Chain Reaction, Plasmid Preparation, Staining, Live Cell Imaging, Electroporation, Expressing

    Analysis of clones from a CANX-mNeonGreen tagging experiment. PCR analysis of single clones using primer for PCR of characteristic fragments indicative for correctly inserted fragments. Primer that anneal to chromosomal DNA were chosen to reside outside of the sequences that are contained in the homology arms for recombination. For western blot analysis antibodies specific to mNeonGreen or to Calnexin were used.
    Figure Legend Snippet: Analysis of clones from a CANX-mNeonGreen tagging experiment. PCR analysis of single clones using primer for PCR of characteristic fragments indicative for correctly inserted fragments. Primer that anneal to chromosomal DNA were chosen to reside outside of the sequences that are contained in the homology arms for recombination. For western blot analysis antibodies specific to mNeonGreen or to Calnexin were used.

    Techniques Used: Clone Assay, Polymerase Chain Reaction, Western Blot

    12) Product Images from "An improved method for high-throughput quantification of autophagy in mammalian cells"

    Article Title: An improved method for high-throughput quantification of autophagy in mammalian cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-68607-w

    Assessing autophagy during viral infection and overexpression/knockout approaches. ( A ), HeLa GL cells were infected with influenza A virus (IAV, left panel, MOI 5), encephalomyocarditis virus (EMCV, middle panel, MOI 10) or measles virus (MeV, right panel, MOI 5). Cells were harvested, saponin treated, fixed, and the eGFP-LC3B fluorescence analyzed by flow cytometry at the indicated time points post infection. Treatment with Chloroquine (1 µM, 4 h) and Rapamycin (1 µM, 4 h) served as controls. Data are shown as mean(n = 3–6) ± SEM. ( B ), HEK293T GL cells were transiently transfected with an empty vector or a TRIM32-FLAG expressing construct. Cells were saponin treated, fixed, and stained with anti-FLAG antibodies (APC) (left and middle panel). eGFP-LC3B MFI of the transfected cell population was quantified for the TRIM32-FLAG sample and background (= vector) subtracted (right panel). Data are shown as mean(n = 3) ± SEM. ( C ), Agarose gel depicting the sgRNA amplicon amplified by PCR from genomic DNA isolated from saponin treated Jurkat GL cells that were either mock-infected or transduced with the Human CRISPR Knockout Pooled Library (GeCKO v2) ( D ) Exemplary distribution of high/low/medium autophagosome content (= eGFP MFI) in Jurkat eGFP-LC3B cells 10 days after transduction with the Human CRISPR Knockout Pooled Library (GeCKO v2) as assessed by FACS sorting ( E ) Scatter Plot (control population ‘input’ vs low autophagy population ‘low’) of aggregated counts of sgRNAs targeting indicated ATG proteins. Each dot is derived from 6 individual sgRNAs targeting the same gene. The dotted red line indicates an equal abundance of sgRNA counts in both populations. Statistical significance was assessed using unpaired t-test ( A ) or one-way ANOVA ( B ). ns = not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Uncropped agarose gel in Supplementary Fig. 5.
    Figure Legend Snippet: Assessing autophagy during viral infection and overexpression/knockout approaches. ( A ), HeLa GL cells were infected with influenza A virus (IAV, left panel, MOI 5), encephalomyocarditis virus (EMCV, middle panel, MOI 10) or measles virus (MeV, right panel, MOI 5). Cells were harvested, saponin treated, fixed, and the eGFP-LC3B fluorescence analyzed by flow cytometry at the indicated time points post infection. Treatment with Chloroquine (1 µM, 4 h) and Rapamycin (1 µM, 4 h) served as controls. Data are shown as mean(n = 3–6) ± SEM. ( B ), HEK293T GL cells were transiently transfected with an empty vector or a TRIM32-FLAG expressing construct. Cells were saponin treated, fixed, and stained with anti-FLAG antibodies (APC) (left and middle panel). eGFP-LC3B MFI of the transfected cell population was quantified for the TRIM32-FLAG sample and background (= vector) subtracted (right panel). Data are shown as mean(n = 3) ± SEM. ( C ), Agarose gel depicting the sgRNA amplicon amplified by PCR from genomic DNA isolated from saponin treated Jurkat GL cells that were either mock-infected or transduced with the Human CRISPR Knockout Pooled Library (GeCKO v2) ( D ) Exemplary distribution of high/low/medium autophagosome content (= eGFP MFI) in Jurkat eGFP-LC3B cells 10 days after transduction with the Human CRISPR Knockout Pooled Library (GeCKO v2) as assessed by FACS sorting ( E ) Scatter Plot (control population ‘input’ vs low autophagy population ‘low’) of aggregated counts of sgRNAs targeting indicated ATG proteins. Each dot is derived from 6 individual sgRNAs targeting the same gene. The dotted red line indicates an equal abundance of sgRNA counts in both populations. Statistical significance was assessed using unpaired t-test ( A ) or one-way ANOVA ( B ). ns = not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Uncropped agarose gel in Supplementary Fig. 5.

    Techniques Used: Infection, Over Expression, Knock-Out, Fluorescence, Flow Cytometry, Transfection, Plasmid Preparation, Expressing, Construct, Staining, Agarose Gel Electrophoresis, Amplification, Polymerase Chain Reaction, Isolation, Transduction, CRISPR, FACS, Derivative Assay

    13) Product Images from "A comprehensive analysis of e-CAS cell line reveals they are mouse macrophages"

    Article Title: A comprehensive analysis of e-CAS cell line reveals they are mouse macrophages

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26512-3

    PCR reactions for equine TLR s. Reactions performed with ( A ), DNA sample obtained from e-CAS cells and ( B ), DNA sample obtained from eqT8888 cells. L: Ladder. The picture has been cropped for clarity and conciseness. A picture of the full-length gel is presented in Supplementary Fig. S1 .
    Figure Legend Snippet: PCR reactions for equine TLR s. Reactions performed with ( A ), DNA sample obtained from e-CAS cells and ( B ), DNA sample obtained from eqT8888 cells. L: Ladder. The picture has been cropped for clarity and conciseness. A picture of the full-length gel is presented in Supplementary Fig. S1 .

    Techniques Used: Polymerase Chain Reaction

    14) Product Images from "An advanced genetic toolkit for exploring the biology of the rock-inhabiting black fungus Knufia petricola"

    Article Title: An advanced genetic toolkit for exploring the biology of the rock-inhabiting black fungus Knufia petricola

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-79120-5

    Generation of marker-free mutants with the CRISPR/Cas9 technique. ( a ) Strategies for CRISPR/Cas9-assisted inactivation of pks1 . Three protospacers (PS) for inducing DSBs in different regions of pks1 were combined with the sgRNA backbone, cas9 , a hygR cassette as well as the AMA1 sequence (Table S1 ). PS- and PS-adjacent motifs (PAM) are shown. ( b ) Random mutation of pks1 via non-homologous end joining (NHEJ). WT:A95 protoplasts were transformed with the circular cas9/pks1 -sgRNA-containing AMA plasmids (Table S3 ). 31 mel− mutants were studied. MEA was inoculated with cell suspensions and incubated for 11 days. ∆ p/ ∆ p − ∆ pks1/∆phd1 . Sequencing of PS-spanning regions revealed point mutations and short deletions at the Cas9 sites (green triangles) resulting in truncated proteins. Mutants H2.7 and H2.6 contain in-frame deletions (Fig. S5 ). ( c ) Increase of editing efficiency by addition of single-stranded DNA oligonucleotides. WT:A95 protoplasts were co-transformed with cas9 -sgRNA-containing AMA plasmids and single-stranded 80-bp-long DNA oligonucleotides (+ O) that covered the Cas9 cutting sites and comprised mutations (Fig. S6 ). Numbers of differentially pigmented colonies on the transformation plates were quantified from four independent experiments (Table S6 ). Representative plates for pks1 are shown on the left. The PS-spanning regions of chosen mutants were amplified by PCR and sequenced to detect the mutations at the Cas9 cutting sites (Fig. S6 ). MEA was inoculated with cell suspensions and incubated for 8 days.
    Figure Legend Snippet: Generation of marker-free mutants with the CRISPR/Cas9 technique. ( a ) Strategies for CRISPR/Cas9-assisted inactivation of pks1 . Three protospacers (PS) for inducing DSBs in different regions of pks1 were combined with the sgRNA backbone, cas9 , a hygR cassette as well as the AMA1 sequence (Table S1 ). PS- and PS-adjacent motifs (PAM) are shown. ( b ) Random mutation of pks1 via non-homologous end joining (NHEJ). WT:A95 protoplasts were transformed with the circular cas9/pks1 -sgRNA-containing AMA plasmids (Table S3 ). 31 mel− mutants were studied. MEA was inoculated with cell suspensions and incubated for 11 days. ∆ p/ ∆ p − ∆ pks1/∆phd1 . Sequencing of PS-spanning regions revealed point mutations and short deletions at the Cas9 sites (green triangles) resulting in truncated proteins. Mutants H2.7 and H2.6 contain in-frame deletions (Fig. S5 ). ( c ) Increase of editing efficiency by addition of single-stranded DNA oligonucleotides. WT:A95 protoplasts were co-transformed with cas9 -sgRNA-containing AMA plasmids and single-stranded 80-bp-long DNA oligonucleotides (+ O) that covered the Cas9 cutting sites and comprised mutations (Fig. S6 ). Numbers of differentially pigmented colonies on the transformation plates were quantified from four independent experiments (Table S6 ). Representative plates for pks1 are shown on the left. The PS-spanning regions of chosen mutants were amplified by PCR and sequenced to detect the mutations at the Cas9 cutting sites (Fig. S6 ). MEA was inoculated with cell suspensions and incubated for 8 days.

    Techniques Used: Marker, CRISPR, Sequencing, Mutagenesis, Non-Homologous End Joining, Transformation Assay, Microelectrode Array, Incubation, Amplification, Polymerase Chain Reaction

    15) Product Images from "EpicPCR 2.0: Technical and Methodological Improvement of a Cutting-Edge Single-Cell Genomic Approach"

    Article Title: EpicPCR 2.0: Technical and Methodological Improvement of a Cutting-Edge Single-Cell Genomic Approach

    Journal: Microorganisms

    doi: 10.3390/microorganisms9081649

    Efficiencies of two- and three-steps epicPCR protocols using different blocking primers (BPs) concentrations. The epicPCRs were run on SXT/R391 carrying bacteria from the Meurthe River water. A and C indicate wells with amplification products from two-steps epicPCR protocols (fusion-PCR on polyacrylamide beads + nested PCR). In the A lane, fusion-PCR products from the first step were used as template DNA in the second step without dilution whereas these products were diluted ten times in line C to circumvent the possible presence of PCR inhibitors. B indicates wells loaded with amplification products resulting from a three-steps epicPCR protocol (fusion PCR on polyacrylamide beads + blocking PCR with BPs as sole primers + nested PCR). The expected size of the final nested-PCR product is around 350 bp (depending on the 16S rRNA gene fragment polymorphism). The conditions of epicPCR as used in Hultman et al. (2018) and that we determined to be the best in our conditions (epicPCR 2.0) are indicated by black arrows. The minus symbols indicate negative controls with epicPCRs run without polyacrylamide beads-template.
    Figure Legend Snippet: Efficiencies of two- and three-steps epicPCR protocols using different blocking primers (BPs) concentrations. The epicPCRs were run on SXT/R391 carrying bacteria from the Meurthe River water. A and C indicate wells with amplification products from two-steps epicPCR protocols (fusion-PCR on polyacrylamide beads + nested PCR). In the A lane, fusion-PCR products from the first step were used as template DNA in the second step without dilution whereas these products were diluted ten times in line C to circumvent the possible presence of PCR inhibitors. B indicates wells loaded with amplification products resulting from a three-steps epicPCR protocol (fusion PCR on polyacrylamide beads + blocking PCR with BPs as sole primers + nested PCR). The expected size of the final nested-PCR product is around 350 bp (depending on the 16S rRNA gene fragment polymorphism). The conditions of epicPCR as used in Hultman et al. (2018) and that we determined to be the best in our conditions (epicPCR 2.0) are indicated by black arrows. The minus symbols indicate negative controls with epicPCRs run without polyacrylamide beads-template.

    Techniques Used: Blocking Assay, Amplification, Polymerase Chain Reaction, Nested PCR

    Control epicPCR amplifications targeting SXT/R391 ICEs performed on beads carrying E. coli MG1656::SXT MO10 as template. Each experiment was done in duplicates with a no-template DNA control, using either the Phusion DNA Polymerase GC or HF buffers. In the nested-PCR step, the use of blocking primers (BPs) has been done as depicted in Spencer et al. (2016), and usually performed so far. All these conditions are summarized in the table upper the gel. The expected size of the desired DNA fragment is 367 bp. Black arrows indicate epicPCR products obtained after performing fusion and nested PCRs using HF but not GC buffer.
    Figure Legend Snippet: Control epicPCR amplifications targeting SXT/R391 ICEs performed on beads carrying E. coli MG1656::SXT MO10 as template. Each experiment was done in duplicates with a no-template DNA control, using either the Phusion DNA Polymerase GC or HF buffers. In the nested-PCR step, the use of blocking primers (BPs) has been done as depicted in Spencer et al. (2016), and usually performed so far. All these conditions are summarized in the table upper the gel. The expected size of the desired DNA fragment is 367 bp. Black arrows indicate epicPCR products obtained after performing fusion and nested PCRs using HF but not GC buffer.

    Techniques Used: Nested PCR, Blocking Assay

    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: 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

    18) 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

    19) 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

    20) Product Images from "Selective sequestration of signaling proteins in a membraneless organelle reinforces the spatial regulation of asymmetry in Caulobacter crescentus"

    Article Title: Selective sequestration of signaling proteins in a membraneless organelle reinforces the spatial regulation of asymmetry in Caulobacter crescentus

    Journal: Nature microbiology

    doi: 10.1038/s41564-019-0647-7

    High throughput assay to probe gene expression as a function of genomic position. a. Graphical representation of the BacTRIP method. (I) A library of reporter plasmids generates a library of barcoded Caulobacter loci (purple, green, and red). Light gray: Tn5 mediated transposon. This includes a 5’ mosaic end (ME) followed by a SalI site, kanamycin resistance cassette (KanR), the origin of replication ( ori ), a promoter (gray), a gene of interest (yellow), a transcribed barcode (colors), a stop codon, the metK gene terminator from Caulobacter , and the 3’ ME. Transformation of the reporter plasmid library into Caulobacter introduces at most one barcode into each cell at a random locus (light brown box). Each barcoded strain reports local transcriptional activity at its locus. (II) DNA. Genomic DNA is used both to determine the integration locus (left) and the number of times each barcode was integrated (right). To determine the locus of each integrated barcode we digested the extracted DNA with SalI and used reverse PCR with mapping primers ( Supplementary Table 8 ) to amplify fragments with an ori element for sequencing. To determine the number of times each barcode was integrated we amplified and sequenced the extracted DNA with counting primers ( Supplementary Table 8 ). (III) RNA. We produced cDNA for sequencing from extracted, reverse-transcribed mRNA. The resulting quantity of inferred mRNA abundance was normalized by total copies of the barcode present in the library. b. BacTRIP results. We used three different CtrA binding sites to probe the wildtype CCNA_00350 promoter (P WT350 ), an engineered promoter in which we replaced the CtrA binding motif (red) of P 350 with a consensus motif (P AT350 ), as well as a mutant site that is not recognized by CtrA (P MT350 ). We synchronized the three populations of cells and preformed BacTRIP on predivisional cells at the 90 minute time point. The bar graphs show abundance of 161, 156, and 148 barcoded eyfp as a function of locus distance from the new pole for pTripTn5- P WT350 :: eyfp , pTripTn5- P AT350 :: eyfp , and pTripTn5- P MT350 :: eyfp .
    Figure Legend Snippet: High throughput assay to probe gene expression as a function of genomic position. a. Graphical representation of the BacTRIP method. (I) A library of reporter plasmids generates a library of barcoded Caulobacter loci (purple, green, and red). Light gray: Tn5 mediated transposon. This includes a 5’ mosaic end (ME) followed by a SalI site, kanamycin resistance cassette (KanR), the origin of replication ( ori ), a promoter (gray), a gene of interest (yellow), a transcribed barcode (colors), a stop codon, the metK gene terminator from Caulobacter , and the 3’ ME. Transformation of the reporter plasmid library into Caulobacter introduces at most one barcode into each cell at a random locus (light brown box). Each barcoded strain reports local transcriptional activity at its locus. (II) DNA. Genomic DNA is used both to determine the integration locus (left) and the number of times each barcode was integrated (right). To determine the locus of each integrated barcode we digested the extracted DNA with SalI and used reverse PCR with mapping primers ( Supplementary Table 8 ) to amplify fragments with an ori element for sequencing. To determine the number of times each barcode was integrated we amplified and sequenced the extracted DNA with counting primers ( Supplementary Table 8 ). (III) RNA. We produced cDNA for sequencing from extracted, reverse-transcribed mRNA. The resulting quantity of inferred mRNA abundance was normalized by total copies of the barcode present in the library. b. BacTRIP results. We used three different CtrA binding sites to probe the wildtype CCNA_00350 promoter (P WT350 ), an engineered promoter in which we replaced the CtrA binding motif (red) of P 350 with a consensus motif (P AT350 ), as well as a mutant site that is not recognized by CtrA (P MT350 ). We synchronized the three populations of cells and preformed BacTRIP on predivisional cells at the 90 minute time point. The bar graphs show abundance of 161, 156, and 148 barcoded eyfp as a function of locus distance from the new pole for pTripTn5- P WT350 :: eyfp , pTripTn5- P AT350 :: eyfp , and pTripTn5- P MT350 :: eyfp .

    Techniques Used: High Throughput Screening Assay, Expressing, Transformation Assay, Plasmid Preparation, Activity Assay, Polymerase Chain Reaction, Sequencing, Amplification, Produced, Binding Assay, Mutagenesis

    21) 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: PLoS Genetics

    doi: 10.1371/journal.pgen.1008647

    (A) Assay to detect CRISPR/Cas9-mediated cleavage in vitro . The region of the Muc14a gene that was amplified contains 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 βtub85D-cas9 transgene (1X), two copies of βtub85D-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 βtub85D-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/βtub85D-cas9 males crossed to w females compared to w and βtub85D-cas9/+ control males crossed to w females. Left columns: embryos to pupae survival rate; central columns: pupae to adults survival rate and right columns: fraction of males and females in adults. 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 . The region of the Muc14a gene that was amplified contains 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 βtub85D-cas9 transgene (1X), two copies of βtub85D-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 βtub85D-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/βtub85D-cas9 males crossed to w females compared to w and βtub85D-cas9/+ control males crossed to w females. Left columns: embryos to pupae survival rate; central columns: pupae to adults survival rate and right columns: fraction of males and females in adults. 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, Amplification, Binding Assay, Polymerase Chain Reaction, Multiplex Assay

    22) 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

    23) Product Images from "A comprehensive analysis of e-CAS cell line reveals they are mouse macrophages"

    Article Title: A comprehensive analysis of e-CAS cell line reveals they are mouse macrophages

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26512-3

    PCR reactions for equine TLR s. Reactions performed with ( A ), DNA sample obtained from e-CAS cells and ( B .
    Figure Legend Snippet: PCR reactions for equine TLR s. Reactions performed with ( A ), DNA sample obtained from e-CAS cells and ( B .

    Techniques Used: Polymerase Chain Reaction

    24) Product Images from "Development of a recombinase polymerase amplification combined with lateral-flow dipstick assay for detection of bovine ephemeral fever virus"

    Article Title: Development of a recombinase polymerase amplification combined with lateral-flow dipstick assay for detection of bovine ephemeral fever virus

    Journal: Molecular and Cellular Probes

    doi: 10.1016/j.mcp.2017.12.003

    Analytical specificity of the BEFV LFD-RPA assay. (A) Specificity of the LFD-RPA assay. The specificity of the assay was assessed for other bovine viral pathogens with similar clinic and etiologies. Lane 1: positive control of BEFV; Lanes 2 to 7: BVDV, IBRV, BPIV-3, BRSV, BcoV and VSV, respectively. Samples were tested in triplicate with one reaction displayed in figure for each triplicate. (B) The results of amplification products of the LFD-RPA on 2% agarose gel. Lane 1: positive control of BEFV; Lanes 2 to 7: BVDV, IBRV, BPIV-3, BRSV, BcoV and VSV, respectively. (C) The quality detection of RNA/DNA of BVDV, IBRV, BPIV-3, BRSV, BcoV and VSV. The RNA/DNA of BEFV, BVDV, IBRV, BPIV-3, BRSV, BcoV and VSV prepared for specificity detection were undertook PCR reaction with viral specific primers ( Supplementary Table 1 ). The positive amplification results were shown in Lane 1, Lane 3, Lane 5, Lane 7, Lane 9, Lane 11, Lane 13, respectively. Lane 2, Lane 4, Lane 6, Lane 8, Lane 10, Lane 12, Lane 14 were negative controls with DNase-free water as template.
    Figure Legend Snippet: Analytical specificity of the BEFV LFD-RPA assay. (A) Specificity of the LFD-RPA assay. The specificity of the assay was assessed for other bovine viral pathogens with similar clinic and etiologies. Lane 1: positive control of BEFV; Lanes 2 to 7: BVDV, IBRV, BPIV-3, BRSV, BcoV and VSV, respectively. Samples were tested in triplicate with one reaction displayed in figure for each triplicate. (B) The results of amplification products of the LFD-RPA on 2% agarose gel. Lane 1: positive control of BEFV; Lanes 2 to 7: BVDV, IBRV, BPIV-3, BRSV, BcoV and VSV, respectively. (C) The quality detection of RNA/DNA of BVDV, IBRV, BPIV-3, BRSV, BcoV and VSV. The RNA/DNA of BEFV, BVDV, IBRV, BPIV-3, BRSV, BcoV and VSV prepared for specificity detection were undertook PCR reaction with viral specific primers ( Supplementary Table 1 ). The positive amplification results were shown in Lane 1, Lane 3, Lane 5, Lane 7, Lane 9, Lane 11, Lane 13, respectively. Lane 2, Lane 4, Lane 6, Lane 8, Lane 10, Lane 12, Lane 14 were negative controls with DNase-free water as template.

    Techniques Used: Recombinase Polymerase Amplification, Positive Control, Amplification, Agarose Gel Electrophoresis, Polymerase Chain Reaction

    25) Product Images from "Fungus-originated genes in the genomes of cereal and pasture grasses acquired through ancient lateral transfer"

    Article Title: Fungus-originated genes in the genomes of cereal and pasture grasses acquired through ancient lateral transfer

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-76478-4

    PCR-based detection of Lp FTRL-related gene from retail products. ( a ) Retail products of barley (i), wheat (ii), oat (iii), and rice (iv). ( b ) PCR assay results were visualised on an agarose gel, using the primers designed for Lp FTRL. The size (bp) of PCR amplicons is indicated on the right side of each image. As a control experiment, PCR primers for the florigen candidate gene (FT in wheat and barley, and Hd3a in rice) were used. For demonstration of absence of Epichloë and Claviceps species in the retail products, PCR primers specific to the fungal species (EfMCF_F and R, and C.purpurea_D0288F and D0289R) were used. The gDNA samples from the perennial ryegrass genotype Impact 04 and E. festucae were used, as positive controls for amplification with the plant and fungus-specific primers, respectively. With the PCR primers specific to Claviceps species, amplification from E. festucae gDNA template was observed, presumably due to sequence similarity between Epichloë and Claviceps species. ‘NTC’ stands for ‘no DNA template control’.
    Figure Legend Snippet: PCR-based detection of Lp FTRL-related gene from retail products. ( a ) Retail products of barley (i), wheat (ii), oat (iii), and rice (iv). ( b ) PCR assay results were visualised on an agarose gel, using the primers designed for Lp FTRL. The size (bp) of PCR amplicons is indicated on the right side of each image. As a control experiment, PCR primers for the florigen candidate gene (FT in wheat and barley, and Hd3a in rice) were used. For demonstration of absence of Epichloë and Claviceps species in the retail products, PCR primers specific to the fungal species (EfMCF_F and R, and C.purpurea_D0288F and D0289R) were used. The gDNA samples from the perennial ryegrass genotype Impact 04 and E. festucae were used, as positive controls for amplification with the plant and fungus-specific primers, respectively. With the PCR primers specific to Claviceps species, amplification from E. festucae gDNA template was observed, presumably due to sequence similarity between Epichloë and Claviceps species. ‘NTC’ stands for ‘no DNA template control’.

    Techniques Used: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Amplification, Sequencing

    26) Product Images from "Microbial single-strand annealing proteins enable CRISPR gene-editing tools with improved knock-in efficiencies and reduced off-target effects"

    Article Title: Microbial single-strand annealing proteins enable CRISPR gene-editing tools with improved knock-in efficiencies and reduced off-target effects

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa1264

    Test template design guideline, junction precision, and capacity of REDIT gene-editing methods. ( A ) Homology arm (HA) length test comparing different template designs of HDR donors (longer HAs) or NHEJ/MMEJ donors (zero/shorter HAs) using REDIT and Cas9 references. Top and bottom are two genomic loci tested using mKate knock-in assay. ( B ) Design of junction profiling assay through isolation of knock-in clones, followed by genomic PCR using primers (fwd, rev) binding outside donor to avoid template amplification. Paired Sanger sequencing of the PCR products reveal homologous and non-homologous edits at the 5′- and 3′- junctions. ( C ) Percentage of colonies with indicated junction profiles from the Sanger sequencing of knock-in clones as in panel B. Indels are insertions or deletions detected from sequencing of ∼48 colonies per condition per locus. Editing methods and donor DNA listed at the bottom (HA lengths indicated in bracket). ( D ) Knock-in efficiencies using a 2-kb cassette to insert dual-GFP/mKate tags to validate REDIT methods with Cas9 references. HA lengths of donor DNAs indicated at the bottom.
    Figure Legend Snippet: Test template design guideline, junction precision, and capacity of REDIT gene-editing methods. ( A ) Homology arm (HA) length test comparing different template designs of HDR donors (longer HAs) or NHEJ/MMEJ donors (zero/shorter HAs) using REDIT and Cas9 references. Top and bottom are two genomic loci tested using mKate knock-in assay. ( B ) Design of junction profiling assay through isolation of knock-in clones, followed by genomic PCR using primers (fwd, rev) binding outside donor to avoid template amplification. Paired Sanger sequencing of the PCR products reveal homologous and non-homologous edits at the 5′- and 3′- junctions. ( C ) Percentage of colonies with indicated junction profiles from the Sanger sequencing of knock-in clones as in panel B. Indels are insertions or deletions detected from sequencing of ∼48 colonies per condition per locus. Editing methods and donor DNA listed at the bottom (HA lengths indicated in bracket). ( D ) Knock-in efficiencies using a 2-kb cassette to insert dual-GFP/mKate tags to validate REDIT methods with Cas9 references. HA lengths of donor DNAs indicated at the bottom.

    Techniques Used: Non-Homologous End Joining, Knock-In, Isolation, Clone Assay, Polymerase Chain Reaction, Binding Assay, Amplification, Sequencing

    27) 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

    28) Product Images from "CRISPR-Cas12a–assisted PCR tagging of mammalian genes"

    Article Title: CRISPR-Cas12a–assisted PCR tagging of mammalian genes

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201910210

    Analysis of clones from a CANX -mNeonGreen tagging experiment. PCR analysis of single clones using primer for PCR of characteristic fragments indicative for correctly inserted fragments. Primers that anneal to chromosomal DNA were chosen to reside outside of the sequences that are contained in the homology arms for recombination. For Western blot analysis, antibodies specific to mNeonGreen or to Calnexin were used.
    Figure Legend Snippet: Analysis of clones from a CANX -mNeonGreen tagging experiment. PCR analysis of single clones using primer for PCR of characteristic fragments indicative for correctly inserted fragments. Primers that anneal to chromosomal DNA were chosen to reside outside of the sequences that are contained in the homology arms for recombination. For Western blot analysis, antibodies specific to mNeonGreen or to Calnexin were used.

    Techniques Used: Clone Assay, Polymerase Chain Reaction, Western Blot

    Tagging efficiency as a function of different parameters. (a) Length of homology arms. M1 and M2 tagging oligos containing the indicated sequence lengths of homology arm (5′-HA and 3′-HA, respectively) to the destination locus were used for PCR tagging of the HNRNPA1 locus in HEK293T cells. Tagging efficiency was estimated 3 d after transfection as described before. Data from three replicates are shown. Error bars indicate SD. (b) PCR cassettes containing various types of ends to direct the choice of DNA repair pathway: homology arms (90-bp and 55-bp homology, for HR; A), blunt ended arms without homology to the target locus (blunt; B), HgaI cut (D), and uncut ends (C). Cutting with the type IIS restriction enzyme HgaI results in 5-nt 3′ overhangs that are complementary to the overhangs generated by the crRNA directed Cas12a-cleavage of the destination locus. Tagging efficiency was estimated 3 d later as described in panel a using HEK293T cells. Data from three replicates are shown. Error bars indicate SD. (c) Use of modified and/or purified oligos. M1/M2 tagging oligos with the indicated number of phosphorothioate bonds and/or biotin as indicated were used for generation of PCR cassettes. All oligos were cartridge purified except for the ones denoted with PAGE, which were size selected using PAGE. Tagging efficiency was estimated 3 d after transfection as described before using HEK293T cells. Data from three replicates are shown. Error bars indicate SD.
    Figure Legend Snippet: Tagging efficiency as a function of different parameters. (a) Length of homology arms. M1 and M2 tagging oligos containing the indicated sequence lengths of homology arm (5′-HA and 3′-HA, respectively) to the destination locus were used for PCR tagging of the HNRNPA1 locus in HEK293T cells. Tagging efficiency was estimated 3 d after transfection as described before. Data from three replicates are shown. Error bars indicate SD. (b) PCR cassettes containing various types of ends to direct the choice of DNA repair pathway: homology arms (90-bp and 55-bp homology, for HR; A), blunt ended arms without homology to the target locus (blunt; B), HgaI cut (D), and uncut ends (C). Cutting with the type IIS restriction enzyme HgaI results in 5-nt 3′ overhangs that are complementary to the overhangs generated by the crRNA directed Cas12a-cleavage of the destination locus. Tagging efficiency was estimated 3 d later as described in panel a using HEK293T cells. Data from three replicates are shown. Error bars indicate SD. (c) Use of modified and/or purified oligos. M1/M2 tagging oligos with the indicated number of phosphorothioate bonds and/or biotin as indicated were used for generation of PCR cassettes. All oligos were cartridge purified except for the ones denoted with PAGE, which were size selected using PAGE. Tagging efficiency was estimated 3 d after transfection as described before using HEK293T cells. Data from three replicates are shown. Error bars indicate SD.

    Techniques Used: Sequencing, Polymerase Chain Reaction, Transfection, Generated, Modification, Purification, Polyacrylamide Gel Electrophoresis

    PCR strategy. PCR is performed with M1 and M2 tagging oligos and a template cassette that contains the tag. The M1 and M2 tagging oligos provide the homology arms (HA; ∼55–90 nt in length) for targeted integration. The M2 tagging oligo additionally provides the direct repeat and a protospacer sequence (orange) for a Cas12a endonuclease. The template cassette contains the desired tag and additional features, such as a selection marker. It also contains the U6 Pol III promoter for driving crRNA expression. PCR yields a linear DNA fragment (PCR cassette) that contains homology arms to the target locus and a functional crRNA gene to cleave the locus.
    Figure Legend Snippet: PCR strategy. PCR is performed with M1 and M2 tagging oligos and a template cassette that contains the tag. The M1 and M2 tagging oligos provide the homology arms (HA; ∼55–90 nt in length) for targeted integration. The M2 tagging oligo additionally provides the direct repeat and a protospacer sequence (orange) for a Cas12a endonuclease. The template cassette contains the desired tag and additional features, such as a selection marker. It also contains the U6 Pol III promoter for driving crRNA expression. PCR yields a linear DNA fragment (PCR cassette) that contains homology arms to the target locus and a functional crRNA gene to cleave the locus.

    Techniques Used: Polymerase Chain Reaction, Sequencing, Selection, Marker, Expressing, Functional Assay

    29) 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

    30) Product Images from "Restriction enzyme digestion of host DNA enhances universal detection of parasitic pathogens in blood via targeted amplicon deep sequencing"

    Article Title: Restriction enzyme digestion of host DNA enhances universal detection of parasitic pathogens in blood via targeted amplicon deep sequencing

    Journal: Microbiome

    doi: 10.1186/s40168-018-0540-2

    Reduction of host DNA by restriction enzyme digestion enhances PCR amplification of parasite DNA. DNA extraction from parasite-infected whole blood yields a DNA sample containing high amounts of host DNA (blue) and low amounts of parasite DNA (bright red). a Performing conventional PCR on this sample, using universal primers, amplifies primarily host DNA (blue), and yields sequencing reads almost entirely belonging to the host. b In contrast, restriction enzyme digestion of host DNA prior to PCR alters the ratio of host to parasite DNA in the initial sample, allowing for selective amplification of parasite DNA (bright red) and resulting in an increase in the relative number of parasite amplicons post-PCR and an increase in the sensitivity of parasite detection via NGS
    Figure Legend Snippet: Reduction of host DNA by restriction enzyme digestion enhances PCR amplification of parasite DNA. DNA extraction from parasite-infected whole blood yields a DNA sample containing high amounts of host DNA (blue) and low amounts of parasite DNA (bright red). a Performing conventional PCR on this sample, using universal primers, amplifies primarily host DNA (blue), and yields sequencing reads almost entirely belonging to the host. b In contrast, restriction enzyme digestion of host DNA prior to PCR alters the ratio of host to parasite DNA in the initial sample, allowing for selective amplification of parasite DNA (bright red) and resulting in an increase in the relative number of parasite amplicons post-PCR and an increase in the sensitivity of parasite detection via NGS

    Techniques Used: Polymerase Chain Reaction, Amplification, DNA Extraction, Infection, Sequencing, Next-Generation Sequencing

    31) 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

    32) 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

    33) 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

    34) 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

    35) 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

    36) 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

    37) Product Images from "Restriction enzyme digestion of host DNA enhances universal detection of parasitic pathogens in blood via targeted amplicon deep sequencing"

    Article Title: Restriction enzyme digestion of host DNA enhances universal detection of parasitic pathogens in blood via targeted amplicon deep sequencing

    Journal: Microbiome

    doi: 10.1186/s40168-018-0540-2

    Reduction of host DNA by restriction enzyme digestion enhances PCR amplification of parasite DNA. DNA extraction from parasite-infected whole blood yields a DNA sample containing high amounts of host DNA (blue) and low amounts of parasite DNA (bright red). a Performing conventional PCR on this sample, using universal primers, amplifies primarily host DNA (blue), and yields sequencing reads almost entirely belonging to the host. b In contrast, restriction enzyme digestion of host DNA prior to PCR alters the ratio of host to parasite DNA in the initial sample, allowing for selective amplification of parasite DNA (bright red) and resulting in an increase in the relative number of parasite amplicons post-PCR and an increase in the sensitivity of parasite detection via NGS
    Figure Legend Snippet: Reduction of host DNA by restriction enzyme digestion enhances PCR amplification of parasite DNA. DNA extraction from parasite-infected whole blood yields a DNA sample containing high amounts of host DNA (blue) and low amounts of parasite DNA (bright red). a Performing conventional PCR on this sample, using universal primers, amplifies primarily host DNA (blue), and yields sequencing reads almost entirely belonging to the host. b In contrast, restriction enzyme digestion of host DNA prior to PCR alters the ratio of host to parasite DNA in the initial sample, allowing for selective amplification of parasite DNA (bright red) and resulting in an increase in the relative number of parasite amplicons post-PCR and an increase in the sensitivity of parasite detection via NGS

    Techniques Used: Polymerase Chain Reaction, Amplification, DNA Extraction, Infection, Sequencing, Next-Generation Sequencing

    38) Product Images from "CRISPR-Cas12a-assisted PCR tagging of mammalian genes"

    Article Title: CRISPR-Cas12a-assisted PCR tagging of mammalian genes

    Journal: bioRxiv

    doi: 10.1101/473876

    PCR Strategy PCR is performed with M1 and M2 tagging oligos and a template cassette that contains the tag. The M1 and M2 tagging oligos provide the homology arms (HA, ∼55 to 90 nt in length) for targeted integration. The M2 tagging oligo additionally provides the direct repeat and a protospacer sequence (orange) for a Cas12a endonuclease. The template cassette contains the desired tag and additional features, such as a selection marker. It also contains the U6 Pol III promoter for driving crRNA expression. PCR yields a linear DNA fragment (PCR cassette) that contains homology arms to the target locus and a functional crRNA gene to cleave the locus.
    Figure Legend Snippet: PCR Strategy PCR is performed with M1 and M2 tagging oligos and a template cassette that contains the tag. The M1 and M2 tagging oligos provide the homology arms (HA, ∼55 to 90 nt in length) for targeted integration. The M2 tagging oligo additionally provides the direct repeat and a protospacer sequence (orange) for a Cas12a endonuclease. The template cassette contains the desired tag and additional features, such as a selection marker. It also contains the U6 Pol III promoter for driving crRNA expression. PCR yields a linear DNA fragment (PCR cassette) that contains homology arms to the target locus and a functional crRNA gene to cleave the locus.

    Techniques Used: Polymerase Chain Reaction, Sequencing, Selection, Marker, Expressing, Functional Assay

    Tagging efficiency as a function of different parameters. ( a ) Length of homology arms. M1 and M2 tagging oligos containing the indicated sequence lengths of homology arm (5’-HA and 3’-HA, respectively) to the destination locus were used for PCR tagging of the HNRNPA1 locus in HEK293T cells. Tagging efficiency was estimated 3 days after transfection as described before. Data from three replicates is shown. Error bars indicate SD. ( b ) PCR cassettes containing various types of ends to direct the choice of DNA repair pathway: Homology arms (90-bp and 55-bp homology, for HR; A ), blunt ended arms without homology to the target locus (blunt; B ), Hga I cut ( D ) and uncut ends ( C ). Cutting with the type IIS restriction enzyme Hga I results in 5-nt 3’-overhangs that are complementary to the overhangs generated by the crRNA directed Cas12a-cleavage of the destination locus. Tagging efficiency was estimated three days later as described in (a) using HEK293T cells. Data from three replicates is shown. Error bars indicate SD. ( c ) Use of modified and/or purified oligos. M1/M2 tagging oligos with the indicated number of phosphorothioate bonds and/or biotin as indicated were used for generation of PCR cassettes. All oligos were ‘cartridge’ purified except for the ones denoted with ‘PAGE’, which were size selected using polyacrylamide gel electrophoresis. Tagging efficiency was estimated three days after transfection as described before using HEK293T cells. Data from three replicates is shown. Error bars indicate SD.
    Figure Legend Snippet: Tagging efficiency as a function of different parameters. ( a ) Length of homology arms. M1 and M2 tagging oligos containing the indicated sequence lengths of homology arm (5’-HA and 3’-HA, respectively) to the destination locus were used for PCR tagging of the HNRNPA1 locus in HEK293T cells. Tagging efficiency was estimated 3 days after transfection as described before. Data from three replicates is shown. Error bars indicate SD. ( b ) PCR cassettes containing various types of ends to direct the choice of DNA repair pathway: Homology arms (90-bp and 55-bp homology, for HR; A ), blunt ended arms without homology to the target locus (blunt; B ), Hga I cut ( D ) and uncut ends ( C ). Cutting with the type IIS restriction enzyme Hga I results in 5-nt 3’-overhangs that are complementary to the overhangs generated by the crRNA directed Cas12a-cleavage of the destination locus. Tagging efficiency was estimated three days later as described in (a) using HEK293T cells. Data from three replicates is shown. Error bars indicate SD. ( c ) Use of modified and/or purified oligos. M1/M2 tagging oligos with the indicated number of phosphorothioate bonds and/or biotin as indicated were used for generation of PCR cassettes. All oligos were ‘cartridge’ purified except for the ones denoted with ‘PAGE’, which were size selected using polyacrylamide gel electrophoresis. Tagging efficiency was estimated three days after transfection as described before using HEK293T cells. Data from three replicates is shown. Error bars indicate SD.

    Techniques Used: Sequencing, Polymerase Chain Reaction, Transfection, Generated, Modification, Purification, Polyacrylamide Gel Electrophoresis

    Endogenous C-terminal gene tagging in mammalian cells using PCR tagging. ( a ) For tag insertion before the STOP codon of an ORF, two ‘Gene specific tagging oligos’ (termed M1 and M2) are designed using an online tool ( www.pcr-tagging.com ). A ‘Tagging PCR’ with a generic ‘Template plasmid’ generates the gene-specific ‘PCR cassette’. The ‘Template plasmid’ provides the tag (e.g. a fluorescent protein), a possible selection marker and a Pol III promoter. For gene tagging the ‘PCR cassette’ is transfected into the target cell together with a helper plasmid containing a Cas12a endonuclease gene. This leads to insertion of the PCR cassette into the chromosome, which yields a fusion of the tag (e.g. GFP) with the target gene. ( b ) Tagging Principle: The PCR cassette contains a crRNA sequence that is expressed inside the cell via an U6 promoter (Pol III promoter). The crRNA directs Cas12a (which is expressed from the helper plasmid) to the target locus close to the insertion site. Stimulated by the DSB the linear PCR cassette is then inserted into the genome. The homology arm of the M1 tagging oligo thereby directs in frame fusion of the tag with the target ORF, leading to the expression of a tagged protein from the target locus. Integration leads to destruction of the crRNA target site, thus preventing re-cleavage of the modified locus. ( c ) Efficiency of C-terminal mNeonGreen-tagging for 16 organelle specific genes. For each gene, specific M1/M2 tagging oligos were used to amplify an mNeonGreen containing template plasmid. The resulting PCR cassettes were transfected in HEK293T cells. HOECHST staining of live cells and analysis by fluorescence microscopy was performed three days after transfection. Fractions of cells exhibiting the expected localization or diffuse cytoplasmic green fluorescence are shown. For information on selected genes, see Table S1. Data from one representative experiment is shown. ( d ) Representative images from HEK293T cells 3 days after transfection. mNeonGreen fluorescence and HOECHST staining (DNA) is shown. In addition to the expected localization, cells showing diffuse cytoplasmic fluorescence (arrows) are detected. ( e ) Tagging is specific for the crRNA and guided by the homology arms (HAs). Efficiency of control transfections (see Fig. S2 for representative examples). * in this transfection indicates that a matching combination of crRNA and HAs was used, but the crRNA was expressed from a different PCR fragment. ** indicates that in this case a PCR cassette was used where the crRNA (for CANX) lead to cleavage of a different gene than the one specified by the HAs (HNRNPA1). A small fraction of cells (
    Figure Legend Snippet: Endogenous C-terminal gene tagging in mammalian cells using PCR tagging. ( a ) For tag insertion before the STOP codon of an ORF, two ‘Gene specific tagging oligos’ (termed M1 and M2) are designed using an online tool ( www.pcr-tagging.com ). A ‘Tagging PCR’ with a generic ‘Template plasmid’ generates the gene-specific ‘PCR cassette’. The ‘Template plasmid’ provides the tag (e.g. a fluorescent protein), a possible selection marker and a Pol III promoter. For gene tagging the ‘PCR cassette’ is transfected into the target cell together with a helper plasmid containing a Cas12a endonuclease gene. This leads to insertion of the PCR cassette into the chromosome, which yields a fusion of the tag (e.g. GFP) with the target gene. ( b ) Tagging Principle: The PCR cassette contains a crRNA sequence that is expressed inside the cell via an U6 promoter (Pol III promoter). The crRNA directs Cas12a (which is expressed from the helper plasmid) to the target locus close to the insertion site. Stimulated by the DSB the linear PCR cassette is then inserted into the genome. The homology arm of the M1 tagging oligo thereby directs in frame fusion of the tag with the target ORF, leading to the expression of a tagged protein from the target locus. Integration leads to destruction of the crRNA target site, thus preventing re-cleavage of the modified locus. ( c ) Efficiency of C-terminal mNeonGreen-tagging for 16 organelle specific genes. For each gene, specific M1/M2 tagging oligos were used to amplify an mNeonGreen containing template plasmid. The resulting PCR cassettes were transfected in HEK293T cells. HOECHST staining of live cells and analysis by fluorescence microscopy was performed three days after transfection. Fractions of cells exhibiting the expected localization or diffuse cytoplasmic green fluorescence are shown. For information on selected genes, see Table S1. Data from one representative experiment is shown. ( d ) Representative images from HEK293T cells 3 days after transfection. mNeonGreen fluorescence and HOECHST staining (DNA) is shown. In addition to the expected localization, cells showing diffuse cytoplasmic fluorescence (arrows) are detected. ( e ) Tagging is specific for the crRNA and guided by the homology arms (HAs). Efficiency of control transfections (see Fig. S2 for representative examples). * in this transfection indicates that a matching combination of crRNA and HAs was used, but the crRNA was expressed from a different PCR fragment. ** indicates that in this case a PCR cassette was used where the crRNA (for CANX) lead to cleavage of a different gene than the one specified by the HAs (HNRNPA1). A small fraction of cells (

    Techniques Used: Polymerase Chain Reaction, Plasmid Preparation, Selection, Marker, Transfection, Sequencing, Expressing, Modification, Staining, Fluorescence, Microscopy

    Exploring transfection parameters ( a ) Impact of transfected amounts of DNA on tagging efficiency using HEK293T cells. Transfected amounts of PCR cassette and Cas12a plasmid as indicated. Always 1 µg of DNA was transfected using lipofectamine. pUC18 was used as neutral DNA. Tagging efficiency was determined three days later by HOECHST staining and live cell imaging. Data from one representative experiment is shown. ( b ) HEK293T cells were transfected for 4 hours or overnight using Lipofectamine 2000 or transfected using electroporation, as indicated. Tagging efficiency was determined three days later as described in (a). Data from one representative experiment is shown. ( c ) HEK293T cells were transfected in duplicates by electroporation with Cas12a protein, Cas12a-encoding mRNA or Cas12-encoding plasmid. For protein-based expression 100 ng of PCR cassette while for mRNA and plasmid 1.5 µg PCR cassette were electroporated. Error bars indicate range between the technical duplicates.
    Figure Legend Snippet: Exploring transfection parameters ( a ) Impact of transfected amounts of DNA on tagging efficiency using HEK293T cells. Transfected amounts of PCR cassette and Cas12a plasmid as indicated. Always 1 µg of DNA was transfected using lipofectamine. pUC18 was used as neutral DNA. Tagging efficiency was determined three days later by HOECHST staining and live cell imaging. Data from one representative experiment is shown. ( b ) HEK293T cells were transfected for 4 hours or overnight using Lipofectamine 2000 or transfected using electroporation, as indicated. Tagging efficiency was determined three days later as described in (a). Data from one representative experiment is shown. ( c ) HEK293T cells were transfected in duplicates by electroporation with Cas12a protein, Cas12a-encoding mRNA or Cas12-encoding plasmid. For protein-based expression 100 ng of PCR cassette while for mRNA and plasmid 1.5 µg PCR cassette were electroporated. Error bars indicate range between the technical duplicates.

    Techniques Used: Transfection, Polymerase Chain Reaction, Plasmid Preparation, Staining, Live Cell Imaging, Electroporation, Expressing

    Analysis of clones from a CANX-mNeonGreen tagging experiment. PCR analysis of single clones using primer for PCR of characteristic fragments indicative for correctly inserted fragments. Primer that anneal to chromosomal DNA were chosen to reside outside of the sequences that are contained in the homology arms for recombination. For western blot analysis antibodies specific to mNeonGreen or to Calnexin were used.
    Figure Legend Snippet: Analysis of clones from a CANX-mNeonGreen tagging experiment. PCR analysis of single clones using primer for PCR of characteristic fragments indicative for correctly inserted fragments. Primer that anneal to chromosomal DNA were chosen to reside outside of the sequences that are contained in the homology arms for recombination. For western blot analysis antibodies specific to mNeonGreen or to Calnexin were used.

    Techniques Used: Clone Assay, Polymerase Chain Reaction, Western Blot

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    New England Biolabs monarch pcr dna cleanup kit
    Mutations were detected in FKH domains of FOXP3 transcripts in HCC. A , representative sequencing chromatograms of point mutations. B , representative sequencing chromatograms of the complicated mutation status. Upper left panel , FKH sequences in mRNA. Upper right panel , sequences of the corresponding regions in genomic <t>DNA.</t> Lower panel , sequences of individual TA clones which were generated to examine individual sequences in multiple-mutation bearing <t>PCR</t> products. C , representative immunohistochemical results of FOXP3-positive lymphocyte distribution in tumors and corresponding nontumorous tissues. Black arrow , FOXP3-positive lymphocytes.
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    Mutations were detected in FKH domains of FOXP3 transcripts in HCC. A , representative sequencing chromatograms of point mutations. B , representative sequencing chromatograms of the complicated mutation status. Upper left panel , FKH sequences in mRNA. Upper right panel , sequences of the corresponding regions in genomic DNA. Lower panel , sequences of individual TA clones which were generated to examine individual sequences in multiple-mutation bearing PCR products. C , representative immunohistochemical results of FOXP3-positive lymphocyte distribution in tumors and corresponding nontumorous tissues. Black arrow , FOXP3-positive lymphocytes.

    Journal: The Journal of Biological Chemistry

    Article Title: The FKH domain in FOXP3 mRNA frequently contains mutations in hepatocellular carcinoma that influence the subcellular localization and functions of FOXP3

    doi: 10.1074/jbc.RA120.012518

    Figure Lengend Snippet: Mutations were detected in FKH domains of FOXP3 transcripts in HCC. A , representative sequencing chromatograms of point mutations. B , representative sequencing chromatograms of the complicated mutation status. Upper left panel , FKH sequences in mRNA. Upper right panel , sequences of the corresponding regions in genomic DNA. Lower panel , sequences of individual TA clones which were generated to examine individual sequences in multiple-mutation bearing PCR products. C , representative immunohistochemical results of FOXP3-positive lymphocyte distribution in tumors and corresponding nontumorous tissues. Black arrow , FOXP3-positive lymphocytes.

    Article Snippet: The 1.2-kb PCR products were purified with Monarch PCR & DNA Cleanup Kit (New England Biolabs) and sent to BGI (Shenzhen, China) for sequencing with primer 5′-GTAGCCATGGAAACAGCACA-3′.

    Techniques: Sequencing, Mutagenesis, Clone Assay, Generated, Polymerase Chain Reaction, Immunohistochemistry

    mRNA reporter design and in-cell and in-solution workflows with in-cell polysome validation. (A) Schematic for the 3’ UTR-barcoded mRNA reporter used to screen mRNA performance in a pooled format. The constant regions and barcode, which flank a variable 3’ UTR, were instrumental for amplifying and identifying hundreds of constructs simultaneously in each of the pooled experiments that comprise PERSIST-seq. The DNA templates for full-length mRNAs were synthesized on the Codex platform and amplified in a pooled PCR using primers complementary to the constant region (T7 promoter) preceding the variable 5’ UTR, and to the ‘constant3’ region following the variable 3’ UTR. (B) Summary of the workflow to progress from the individually synthesized DNA templates to the in vitro synthesized mRNA pool of 233 different constructs. We then use the same mRNA pool to screen mRNA performance in a three-pronged set of in-cell and in-solution expression and stability analyses. (C) Quality control of the 233-mRNA pool on a 1.2% formaldehyde (FA) gel stained with ethidium bromide (EtBr) after 3 hrs of in vitro transcription (IVT). The mRNA pool was analyzed before and after capping and polyadenylation. Pooled IVT is equally efficient with the starting template DNA pool with or without PCR-amplification of the DNA template pool. The three major bands corresponding to the three CDS types are indicated. The RiboRuler High Range RNA ladder (Thermo Fisher) is loaded for reference. (D) Polysome fractionation analysis of a transfected mRNA reporter. As an example, the distribution of an mRNA with short scrambled 5’ and 3’ UTRs 6 hrs after transfection into HEK293T cells was compared to the distribution of endogenous human ActB mRNA. RNA was extracted from fractions and quantified by qPCR with a RNA spike-in for normalization. Values are plotted as mRNA normalized per fraction. (E) In-solution RNA degradation strategy of barcoded mRNAs containing CDS variants with hHBB 5’ and 3’ UTRs. The differential degradation of CDS variants depends on their individual CDS structures. mRNA pools are degraded in solution by nucleophilic attack (red circle). After degradation, RT-PCR is performed to selectively amplify mRNAs that remain intact along their full length. Then, the barcode regions of these full-length mRNAs are PCR-amplified, adaptor-ligated, and prepared for Illumina sequencing.

    Journal: bioRxiv

    Article Title: Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics

    doi: 10.1101/2021.03.29.437587

    Figure Lengend Snippet: mRNA reporter design and in-cell and in-solution workflows with in-cell polysome validation. (A) Schematic for the 3’ UTR-barcoded mRNA reporter used to screen mRNA performance in a pooled format. The constant regions and barcode, which flank a variable 3’ UTR, were instrumental for amplifying and identifying hundreds of constructs simultaneously in each of the pooled experiments that comprise PERSIST-seq. The DNA templates for full-length mRNAs were synthesized on the Codex platform and amplified in a pooled PCR using primers complementary to the constant region (T7 promoter) preceding the variable 5’ UTR, and to the ‘constant3’ region following the variable 3’ UTR. (B) Summary of the workflow to progress from the individually synthesized DNA templates to the in vitro synthesized mRNA pool of 233 different constructs. We then use the same mRNA pool to screen mRNA performance in a three-pronged set of in-cell and in-solution expression and stability analyses. (C) Quality control of the 233-mRNA pool on a 1.2% formaldehyde (FA) gel stained with ethidium bromide (EtBr) after 3 hrs of in vitro transcription (IVT). The mRNA pool was analyzed before and after capping and polyadenylation. Pooled IVT is equally efficient with the starting template DNA pool with or without PCR-amplification of the DNA template pool. The three major bands corresponding to the three CDS types are indicated. The RiboRuler High Range RNA ladder (Thermo Fisher) is loaded for reference. (D) Polysome fractionation analysis of a transfected mRNA reporter. As an example, the distribution of an mRNA with short scrambled 5’ and 3’ UTRs 6 hrs after transfection into HEK293T cells was compared to the distribution of endogenous human ActB mRNA. RNA was extracted from fractions and quantified by qPCR with a RNA spike-in for normalization. Values are plotted as mRNA normalized per fraction. (E) In-solution RNA degradation strategy of barcoded mRNAs containing CDS variants with hHBB 5’ and 3’ UTRs. The differential degradation of CDS variants depends on their individual CDS structures. mRNA pools are degraded in solution by nucleophilic attack (red circle). After degradation, RT-PCR is performed to selectively amplify mRNAs that remain intact along their full length. Then, the barcode regions of these full-length mRNAs are PCR-amplified, adaptor-ligated, and prepared for Illumina sequencing.

    Article Snippet: PCR reactions were purified with Monarch PCR & DNA Cleanup Kit (NEB, T1030L).

    Techniques: Construct, Synthesized, Amplification, Polymerase Chain Reaction, In Vitro, Expressing, Staining, Fractionation, Transfection, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Sequencing

    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.

    Journal: bioRxiv

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

    doi: 10.1101/2020.02.02.931477

    Figure Lengend 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.

    Article Snippet: The synthesized DNA template was purified with the use of a Monarch PCR & DNA Cleanup Kit (New England Biolabs, #T1030L).

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